Methods Of Detecting Cancer

ABSTRACT

The present disclosure is directed toward methods and kits for detecting cancer, and in particular breast cancer, in a subject by measuring the levels of at least one of the identified markers, as compared to a control. The expression of the markers in Table 2A is increased in samples from subjects with cancer as compared to the expression level in subjects without cancer and the expression of the markers in Table 2B are decreased in samples from subjects with cancer as compared to the expression level in subjects without cancer. The sample may be lacrimal secretions or eye wash fluid, saliva, or other biological fluids. The kits may include an eye wash kit, collection tubes and protease inhibitors, or protein stabilizers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional patent application No. 61/991,061 filed on 9 May 2014, which is hereby incorporated by reference in its entirety.

BACKGROUND

1. Field of Invention

This present application encompasses proteins and peptide fragments of those proteins produced by proteolytic digestion that are useful for diagnosing or monitoring for the presence of cancer in an individual.

2. Description of the Related Art

Screening mammograms typically have a sensitivity of 75% and specificity of around 98% resulting in a false positive rate of roughly 5% per mammogram

(Brown, Houn, Sickles, & Kessler, 1995; Kolb, Lichy, & Newhouse, 2002; Luftner & Possinger, 2002). Follow up imaging to evaluate false positives costs the US over 4 B with an additional 1.6 B for biopsies alone. In 2010 of the 1.6 M biopsies performed as little as 16% (only 261,000) were found to have cancer (Grady, 2012). The answer to increasing the diagnostic parameters of imaging can be found in the pre and post image diagnostics which focuses on genetic and proteomic information, more specifically, biomarkers (Armstrong, Handorf, Chen, & Bristol Demeter, 2013; Li, Zhang, Rosenzweig, Wang, & Chan, 2002).

Tissue and serum are commonly the most logical place for beginning biomarker research, however the large dynamic range of both mediums makes discovery quite difficult (Schiess, Wollscheid, & Aebersold, 2009). The answers may lie in less complex biological fluids, such as saliva and tears. The use of tears as diagnostic medium is not a novel application as the tear proteome has been extensively investigated previously (Böhm et al., 2012; 2011; Lebrecht, Boehm, Schmidt, Koelbl, & Grus, 2009a; Lebrecht et al., 2009b; Wu & Zhang, 2007). In this application a quantitative assay for the detection of a panel of tear-based biomarkers in response to cancer by triple quadrupole LC mass spectrometry is proposed. From this quantitative information, the framework for a Certified Laboratory Improvement Amendments (CLIA) protocol will be defined.

SUMMARY

Methods of determining whether a subject has cancer are provided herein. The methods include obtaining a sample from the subject and performing steps for or detecting the level of at least one of the markers provided in Table 2A or Table 2B in the sample. The subject is likely to have cancer if the levels of the markers of Table 2A are increased or if the markers in Table 2B are decreased as compared to the levels in a control sample lacking cancer. The sample is optionally lacrimal secretions, such as an ocular wash, saliva or other bodily fluid.

Kits for performing the methods described herein are also provided. The kits may comprise an eye wash solution and collection materials such as tubes. The tube for collection may comprise a protease inhibitor or other protein stabilizing agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of photographs of a NuPAGE showing the proteins collected from each of the pooled ocular wash samples. The lane numbers correspond to pool numbers with the even numbers being breast cancer pools and the odd numbers being control pools.

FIG. 2 is a graph comparing the protein expression in cancer and control samples showing increased expression of several proteins in breast cancer samples as compared to controls based on peak intensity as determined by LC-MS/MS.

FIG. 3 is a graph comparing the protein expression in cancer and control samples showing decreased expression of several proteins in breast cancer samples as compared to controls based on peak intensity as determined by LC-MS/MS.

DETAILED DESCRIPTION

Provided herein are proteins and trypsin produced polypeptides (as defined in Table 2A and 2B in the Examples and the actual trypsin sequences and full length amino acid sequences of the proteins identified as being up regulated and down regulated in cancer samples are provided in Appendix I and Appendix II, respectively) which are shown in the Examples to increase or decrease in biological samples in response to the presence of breast cancer as compared to controls. These proteins and peptides are biomarkers and will be used to determine the disease state of a patient or other subject.

Subjects include humans, domesticated animals such as cats, dogs, cows, pigs or other animals susceptible to cancer. A “patient” indicates a subject who is diagnosed with a disease or with cancer or being tested for having cancer. Thus subject and cancer may be used interchangeably herein. The subjects may be suspected of having cancer, in particular breast cancer. The subjects may have an increased risk of developing breast cancer. For example, the subject may be at increased risk of cancer or suspected of having cancer because of a positive mammography result, by detection of a lump in the breast, testing positive for a gene known to increase the risk of cancer such as BRCA, or already have had a resection, biopsy or other procedure to remove the cancer. The subject may be undergoing or have previously undergone treatment for cancer and the methods and kits herein are used to monitor progression of treatment or alternatively to monitor for recurrence or spread of the cancer. The cancer may be detected as early as stage I or II cancer, but later stages will also be detected.

Also provided herein are methods and kits to collect ocular wash samples for use to determine the expression levels of the identified proteins or polypeptides in lacrimal secretions. In addition, the use of tubes for collection containing protease inhibitor or protein stabilizing agents is covered. The kits further contain buffers or reagents for the elution of breast cancer biomarkers from the eye. The design of devices to collect the applied saline solution from the corner of the exposed ocular surface as well as the packaging of this device together with saline and a pre-prepared sample collection tube are also disclosed.

The methods disclosed herein encompass the use of these breast cancer biomarkers, singly or in multiples, in a CLIA based protocol utilizing a triple quadrupole LC-MS platform, which will be carried out at a centralized laboratory testing facility. The ocular wash samples collected from individuals may be shipped to the testing facility in this embodiment. The identified proteins and their subsequent proteolytic fragments are used for quantitative analysis of diagnostic peptides produced in the triple quad. A threshold value or a relative or actual value in terms of polypeptide concentration directly relating to the polypeptides listed in Tables 2A and 2B can be defined or samples can be compared directly to non-cancerous controls. The quantitative information in report form could be provided to physicians to help in making decisions regarding the pathway of patient care. Physicians may base treatment decisions on these results and the final step may include administration of an appropriate anti-cancer therapeutic to the subject.

In an alternative embodiment, the polypeptides of Tables 2A and 2B may be detected by implementing binding agents (i.e. antibodies, peptoids, coated surfaces) and reagents that accommodate a binding interaction specific to these proteins to produce a reaction which can be quantitated based on production of a detectable signal such as florescence, color change, or UV absorbance. Implementing these components in a cartridge with a partnering reading instrument that could be used at point of care is also provided. Binding agents for these proteins and polypeptides may also be used for detection in a lateral flow device. Thus methods of detecting the level of protein expression in the samples using a binding partner such as an antibody may be used to detect the markers provided herein in an immunoassay.

The immunoassay typically includes contacting a test sample with an antibody that specifically binds to or otherwise recognizes a biomarker, and detecting the presence of a complex of the antibody bound to the biomarker in the sample. The immunoassay procedure may be selected from a wide variety of immunoassay procedures known to the art involving recognition of antibody/antigen complexes, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), and Western blots, and use of multiplex assays, including use of antibody arrays, wherein several desired antibodies are placed on a support, such as a glass bead or plate, and reacted or otherwise contacted with the test sample. Such assays are well-known to the skilled artisan.

The detection of the biomarkers described herein in a sample may be performed in a variety of ways. In one embodiment, the method provides the reverse-transcription of complementary DNAs from mRNAs obtained from the sample. Fluorescent dye-labeled complementary RNAs may be transcribed from complementary DNAs which are then hybridized to the arrays of oligonucleotide probes. The fluorescent color generated by hybridization is read by machine, such as an Agilent Scanner and data are obtained and processed using software, such as Agilent Feature Extraction Software (9.1). Such array based methods include microarray analysis to develop a gene expression profile. As used herein, the term “gene expression profile” refers to the expression levels of mRNAs or proteins of a panel of genes in the subject. As used herein, the term “panel of diagnostic genes” refers to a panel of genes whose expression level can be relied on to diagnose or predict the status of the disease. Included in this panel of genes are those listed in Tables 2A and 2B, as well as any combination thereof, as provided herein. In other embodiments, complementary DNAs are reverse-transcribed from mRNAs obtained from the sample, amplified and simultaneously quantified by real-time PCR, thereby enabling both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific gene product in the complementary DNA sample as well as the original mRNA sample.

The methods of this invention include detecting at least one biomarker. However, any number of biomarkers may be detected. It is preferred that at least two biomarkers are detected in the analysis. However, it is realized that three, four, or more, including all, of the biomarkers described herein may be utilized in the analysis. Thus, not only can one or more markers be detected, one to 40, preferably two to 40, two to 30, two to 20 biomarkers, two to 10 biomarkers, or some other combination, may be detected and analyzed as described herein. In addition, other biomarkers not herein described may be combined with any of the presently disclosed biomarkers to aid in the diagnosis of cancer. Moreover, any combination of the above biomarkers may be detected in accordance with the present invention.

The markers of Table 2A may be increased at least 2 fold, 4 fold, 5 fold, 8 fold, 10 fold or more relative to the level of the marker in the control sample. The markers of Table 2B are decreased at least 1.5 fold, 2 fold, 3 fold, 4 fold or more relative to the level of the marker in the control sample. The control sample may be a sample from a subject that does not have cancer, a pooled sample from subjects that do not have cancer or may be a control or baseline expression level known to be the average expression level of subjects without cancer.

Several terms are used throughout this disclosure and should be defined as commonly used in the art, or as specifically provided herein. As provided herein, mass spectrometry or MS refers to an analytical technique generating electrical or magnetic fields to determine mass-to-charge ratio of peptides and chemical compounds in order to identify or determine peptide sequence and chemical structures. LC-MS/MS spectrometry refers to an analytical technique combining the separation capabilities of high performance liquid chromatography (HPLC) with the mass analysis of mass spectrometry. Triple quadrupole mass spectrometry refers to a tandem mass spectrometer with three ionizing chambers (Q1, Q2, &Q3). This technique allows for target detection of molecules of interest. Ion pairs refers to a parent peptide detected in Q1 in it's doubly or triply charged form and a resulting y or b ion as generated by Q2 and detected in Q3 of a triple quadrupole mass spectrometry instrument. SIS internal peptide refers to a synthesized isotopically-labeled peptide with the same sequence as the peptide to be monitored in Q1 and used as an internal standard for reference to quantify the peptide of interest. The −y ion refers to an ion generated from the c-terminal of a peptide fragment. The −b ion refers to an ion generated from the n-terminal of a peptide fragment. Quantitative Ion refers to the selected highest intensity y or b ion used to determine the quantity of it's parent protein in a biological sample. Qualitative Ion refers to ion/ions chosen to ensure the integrity of the Qualitative ion to selected protein of interest and labeled peptide to selected standards.

CLIA refers to Clinical Laboratory Improvements Amendments which are federal regulatory standards that apply to all clinical laboratory testing preformed on humans in the united states, except clinical trials and basic research. (CLIA related Federal Register and Code of Federal Regulation Announcements). CLIA approved laboratory refers to a clinical lab which preforms laboratory testing on human specimens for diagnosis, prevention, or treatment of disease or impairment and is approved and monitored by an FDA approved regulatory organization. CLIA waived test refers to a clinical laboratory test meeting specific criteria for risk, error and complexity as defined by the Food and Drug Association (FDA).

Point-of-care device refers to an instrument or cartridge available at the location of patient and physician care containing binding agents to a biomarker, or series of biomarkers of interest, and can generate information on the presence, absence, and in some cases concentrations of detected biomarkers. Analyte refers to any measurable biomarker which can be protein, peptide, macromolecule, metabolite, small molecule, or autoantibody. Biological fluid as used herein refers to tears, whole blood, serum, urine, and saliva. Biomarker refers to any substance (e.g. protein, peptide, metabolite, polynucleotide sequence) whose concentration level changes in the body (e.g. increased or decreased) as a result of a disease or condition. Marker and biomarker may be used interchangeably herein.

Lateral flow test refers to a device used to measure the presence of an analyte in a biological fluid using porous paper of sintered polymer. ELISA refers to Enzyme-linked immunosorbent assay which utilizes antibodies to detect the presence and concentration of an analyte of interest. Diagnostic Panel refers to a group of molecules (e.g. proteins or peptides) whose combined concentrations are used to diagnose a disease state. (e.g. cancer). A breast cancer marker refers to a molecule (e.g. protein, peptide, metabolite, polynucleotide sequence) whose concentration level in the body changes (e.g. is increased or decreased) as a result of breast cancer.

In addition to being useful to diagnose cancer and in particular breast cancer in a subject, the kits and methods provided herein may be used to monitor treatment or recurrence of cancer in an individual previously diagnosed with cancer. Thus if the levels of the markers in Table 2A begin to rise or the levels of the markers in Table 2B begin to decrease over time in the same subject after treatment, further chemotherapeutics targeting the cancer may be administered. The methods and kits may also be used to monitor the effectiveness of a chemotherapeutic treatment. In this alternative, the levels of the biomarkers in Table 2A would decrease over time if the treatment regime is effective and either would not change or may increase over time if the treatment regime is not effective in a single subject. The levels of the biomarkers in Table 2B would increase over time during treatment with a therapeutic that is effective and would either not change or decrease over time if the treatment regime is not effective in a single subject.

Treating cancer includes, but is not limited to, reducing the number of cancer cells or the size of a tumor or mass in the subject, reducing progression of a cancer to a more aggressive form, reducing proliferation of cancer cells or reducing the speed of tumor growth, killing of cancer cells, reducing metastasis of cancer cells or reducing the likelihood of recurrence of a cancer in a subject. Treating a subject as used herein refers to any type of treatment that imparts a benefit to a subject afflicted with a disease or at risk of developing the disease, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc.

The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and/or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including.” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of” and “consisting of” those certain elements.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.

The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

EXAMPLES Example 1 Methods for Collecting Ocular Wash Samples

This study was carried out under institutional review board approval and participants were recruited at two clinics based in Arkansas, The Breast Center and Highlands Oncology Group, as well as two clinics based in Washington, PeaceHealth Southwest and PeaceHealth Longview Surgery Center. Inclusion/exclusion criteria used by the clinic for patient selection is given in Table 1.

TABLE 1 Inclusion/Exclusion Criteria for participant selection Individuals who are: Between the ages of 18-100 years of age Presenting for a routine check up Presenting for the evaluation of an abnormal exam or test (mammogram, ultrasound, MRI, PET, ect.) Presenting for the evaluation of a palpable lump or mass Presenting with a mass, pre or post biopsy as long as a portion of mass is remaining Currently have or are in treatment for breast cancer. Individuals who are: <18 years of age or >100 years of age. experiencing a concurrent eye infection or trauma. Currently experiencing acute conjunctivitis Known to have abnormal production of tears (too much or too little)

Ocular wash samples were obtained by rinsing the exposed surface of the eye with Optics Laboratory single use Eye-Cept Rewetting drops. The single use dropper, selected to eliminate contamination, was used to apply approximately five drops of rewetting saline to the outside corner of the eye. After application the solution naturally flowed across the surface of the eye and pooled in the inner corner/duct next to the nose. The solution was then collected by suction using a one mL tuberculine syringe, with no needle attached, and transferred to a pre-labeled 0.5 mL tube with an o-ring screw top cap. The optimal total volume from each collection is approximately 100 μL, however actual volumes can vary. Samples were stored between −20° C. and −80° C. (depending on freezer unit available) within two hours of collection.

Samples collected at participating clinics were retrieved by Ascendant personnel on a weekly basis and transferred on dry ice to Ascendant's laboratory facility. In the case of the Washington based clinics, samples were shipped to Ascendant on dry ice on a monthly basis.

Data collected from the participants included: sex, race, age, previous cancer history, family history of breast cancer, stage of current cancer (I, II, III, IV) tumor size, breast cancer subtype (Ductal Carcinoma In Situ, Invasive Ductal Carcinoma, Invasive Lobular Carcinoma, Lobular Carcinoma In Situ, and Unknown) and tumor grade. A spreadsheet was created to track data and stratify samples based on selected criteria.

Control samples were collected, using the procedure detailed above, from volunteers between the ages of 18-100 who reported they were cancer and mass free as per the inclusion criteria outlined in the IRB approved collection protocol. Exclusion criteria are the same as for the breast cancer patients. All control participants were recruited from the general population; consent and sample collected was performed by Ascendant Diagnostics personnel. Data collected from control participants included: sex, age, race, previous history of breast cancer, family history of breast cancer, and current medications.

All samples in the tear bank were stored at −80° C. and freeze thaw cycles were limited to three times, as protein degradation was observed after three freeze thaw cycles. In some cases samples were aliquoted to minimize freeze thaw cycles further.

Example 2 Methods for Preparation of Sample Pools for LC MS/MS

Eight pooled samples (four breast cancer pools and four control pools) each with a total volume of 300 μL were assembled from banked tear samples for the purpose of label free quantitation using in-gel digestion. All breast cancer ocular wash samples used were taken from individuals with stage I &II breast cancer and were collected prior to treatment. Controls were age matched for accuracy of comparison.

To ensure sample integrity, MALDI-TOF data was collected on aliquots from each of the individual samples, which were included in the pooled samples. Prior to MALDI testing, tear samples were purified using ZipTip_(e18). This procedure serves to remove any contaminates which may be present in the sample and to concentrate the proteins in order to increase ease of detection. A 15 μL aliquot was removed from the freezer and thawed at room temperature for 10 minutes (˜22° C.). The protocol for ZipTip_(e18) was adapted from the user manual supplied by Millipore and a variable pipette with a total volume capacity of 10 μL was used for all sample preparations. The ZipTip_(e18) was equilibrated in a wetting solution of acetonitrile (ACN) 0.1% TFA for 10 cycles (1 cycle involves aspirating 10 μL of solution into the tip and dispensing). Following equilibration, the tip was washed with ddH₂O (0.1% TFA) for 10 cycles. The sample was then loaded for 10 cycles, followed by a wash with ddH₂O (0.1% TFA) for 10 cycles. The load procedure, followed by the wash procedure was carried out a total of five times to ensure maximum protein binding. Bound proteins were eluted in 5 μL of ACN (0.1% TFA) for 20 cycles into a clean tube. The ACN (0.1% TFA) was removed using an eppendorf vacufuge plus for 10 minutes at 45° C. Samples were then reconstituted in 5 μL ddH₂O (0.1% TFA) and spotted onto a ground steel MALDI target. Each sample was spotted a total of three times at 1 μL each time, allowing complete drying of the spot before more material was added. After the final spotting was completely dry, 1 μL of a saturated solution of 40 mgs of Sinapinic Acid matrix prepared in 1 mL of 50:50 solution of ACN/ddH₂O (0.1% TFA) was spotted onto each sample and all samples were allowed to dry completely on the bench top prior to data collection. One microliter of protein standard was added to several locations on the MALDI target as well. The protein standard was spotted only once and followed by addition of the sinapinic acid matrix used for the OW samples.

Data was collected on a Bruker Reflex III MALDI-IOF mass spectrometer in its linear positive mode, as linear mode increases the sensitivity. Acquisition of all spectra was performed both manually and automatically (user unbiased acquisition) using Bruker Daltonics flex Control software. For each spot, MALDI-TOF mass spectra were acquired at least three times, with a total of 200 laser shots accumulated for each run. Shot accumulation was programmed using a fuzzy logic operator to only consider spectra with S/N better than 20 in between m/z 2000-45,000. Sample integrity was evaluated by visual inspection of the generated MALDI-TOF spectrum. High mass peak splitting together with increased quantity of low mass peaks suggest protein degredation has occurred and the sample was not used further.

Total protein content of each pool was determined using a bicinchoninic acid protein assay kit with a 1:20 (v/v) ratio of standard and unknown to working reagent and an incubation time of 30 min at 37° C. To ensure reliable total protein content calculation, a series of dilutions were made for each sample (i.e. 1:2, 1:4, 1:6) and all dilutions were plated in triplicate. A standard curve using diluted albumin (2 mg/ml, 1.5 mg/ml, 1 m/ml, 0.75 mg/ml, 0.5 mg/ml, 0.25 mg/ml 0.125 mg/ml 0.025 mg/ml and 0 mg/ml) was generated and blank subtraction was applied to all standards and unknowns. The protein concentration for each unknown was calculated using a four-parameter fit of the standard curve. Concentrations were multiplied by the dilution factor and averaged to give an accurate total protein content calculation. Assays were only considered valid if the coefficient of variation (% CV) was 15% or below.

Using the total protein content determined by BCA, 25 μg of protein from each pool was loaded onto a NuPAGE Bis-Tris 4-12% gradient separation gel and run using methods standard for an individual skilled in the art as shown in FIG. 1. Following separation of the ocular proteome, between 20-22 slices were cut for each lane and subjected to disulfide reduction using Dithiothreitol, followed by sulfhydryl aklyation using iodoacetemide, and finally trypsin digestion. Specific slice counts for each sample were as follows: Lane 1=20 slices, Lane 2=21 slices, Lane 3=22 slices, Lane 4=21 slices, Lane 5=20 slices, Lane 6=20 slices, Lane 7=21 slices, Lane 8=21 slices.

Example 3 Methods and Results for Label Free Quantitation by LC MS/MS

Twenty μL from each trypsin digestion reaction was loaded onto a nanoAcquity UPLC (Waters) and eluted using a gradient from 3-99% 0.1% formic acid, 75% acetonitrile over 30 minutes. A LTQ Orbitrap Velos (Thermo Scientific) was used for detection of the peptides produced by proteolytic cleavage. Raw data files from the LC-MS/MS analysis were uploaded into the MASCOT database for protein identification using the UniProtKB database, 2 ppm peptide mass tolerance, and 0.5 Da fragment mass tolerance. The output from MASCOT was then uploaded into the software packages Scaffold and MaxQuant for analysis.

Greater than 700 protein hits were identified using this method. In order to isolate potential biomarker candidates, peak intensities for each group (cancer and control) were averaged for each protein and fold change was determined with respect to cancer. In addition a student's T-test was applied to each protein providing a p-value. All proteins with a fold change of greater than 1.5 and a p-value <0.05 were, considered as possible biomarker candidates. P-values and fold changes were assessed on a case by case basis and some proteins with higher p-values were included in the candidate biomarkers list. The list was then narrowed based on biological relevance to breast cancer, other cancer subtypes, and cancer processes. The complete list of candidate biomarkers is given in Tables 2A and 2B and shown in graphic form in FIGS. 2 and 3.

TABLE 2A Biomarkers with an increase expression in cancer as compared to control samples. Protein ID P-Value Fold Change CLEC3B 0.067 No expression in control KLK8 0.07 No expression in control C8A 0.149 No expression in control HRC 0.17 No expression in control KLK13 0.178 No expression in control C7 0.207 No expression in control ALDH1A1 0.24 No expression in control APOL1 0.32 No expression in control MUC-1 0.27 40.6 BLMH 0.212 38.1 SPRR1B 0.117 35.1 SERPINB2 0.11 16.1 Putative uncharacterized 0.165 11.7 protein RAB-30 0.153 11.3 C4A 0.099 9.6 PRDX6 0.14 7.6 CFHR1 0.169 7.4 A1BG 0.11 7.2 GGH 0.14 7.1 EZR 0.066 6.3 SERPINF2 0.16 5.9 HPX 0.1 5.5 CRISP3 0.0238 5.2 CPA4 0.14 4.8 PGLYRP2 0.06 3.9 CASP14 0.068 3.3 Ig Kappa Chain V-III region 0.001 2.6 POM ALB 0.014 2.4 CFH 0.042 2.1 SLC34A2 0.105 29.3

TABLE 2B Biomarkers with a decrease in expression in cancer samples as compared to controls Protein ID P-value Fold Change GAS6 0.045 3.5 CTSL1 0.051 3.4 SFRP1 0.059 3.4 BPI 0.045 2.5 CHID1 0.0546 2.2 MSN 0.0545 2.06 ERAP1 0.014 1.6 QPCT 0.045 1.6 ATRN 0.062 1.6 LTF 0.051 1.5

To further confirm protein identity, the peptide sequences produced by trypsin digestion were mapped back to the original protein sequence. Trypsin products unique to particular proteins were noted, as these sequences have the potential to be used as diagnostic peptides as well as isotopically labeled standards in the final CLIA triple quadrupole mass spectrometry assay. The sequences of the trypsin products and the full-length proteins markers identified in Tables 2A and 2B are provided in Appendix I and Appendix II, respectively.

Example 4 Methods for Schirmer Strip Collections and Processing

Institutional review board approval was obtained for the collection of tears using Schirmer strips. For collection, the rounded tip of the Schirmer strip was folded over at the 0 mm line forming a lip. The folded portion was placed in the lower eyelid of the participant and they were asked to close their eye and keep it in the closed position for a period of 5 minutes. After five minutes the strip was removed and placed in a sterile 1.5 mL pre-labeled snap top tube and placed at −20° C. or −80° C. depending on availability. Collection criteria stated that if the 35 mm mark was reached prior to the five minute time, the strip could be removed.

Data collected from participants included the following, age, sex, race, currently taking birth control or on hormone replacement therapy, ophthamological infections, current or recent chemotherapy treatments, family history of cancer, genetic testing (BRAC1/2) if available, cancer stage, cancer type, hormone receptor status, size of mass, tumor grad, previous history of cancer. A spreadsheet was constructed to house this information and allow for sample stratification based on desired characteristics. Sample total protein content was also entered into the database.

To elute the proteins bound to the Schirmer strip, the strips were first diced and placed in a clean sterile 1.5 mL snap top tube. 200 μL of 1×PBS was added to the diced strip and the sample was incubated at 4° C. with mild shaking overnight. Following elution, the samples were spun briefly to collect the strip fragments at the bottom of the tube, and the supernatant was transferred to a new clean 1.5 mL snap top tube. Total protein content was determined using BCA assay, as described above, and the samples were stored at −80° C. until further use.

REFERENCES

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APPENDIX I

Sequences shown to be up regulated in subjects with cancer:

Protein Name Uniprot IPI Gene Name: Complement C4A POCOL4 IPI00032258 C4A_Human Trypsin Fragments  1. AEFQDALEK  2. DHAVDLIQK  3. DKGQAGLQR  4. EMSGSPASG  5. FACYYPR  6. FGLLDEDGK     IPVK     K  7. GHLFLQTDQ  8. GLCVATPVQ  9. GIQDEDGYR     PIYNPGQR     LR 10. GPEVQUVAH 11. GSFEFPVGD 12. HLVPGAPFL     SPWLK     AVSK     LQALVR 13. LLATLCSAE 14. LNMGITDLQ 15. ITQVLHFTK     VCQCAEGK     GLR      16. NVNFQK 17. QGSFQGGFR 18. SCGLHQLLR      19. VDFTLSSER 20. VDVQAGACE 21. VFALDQK     GK 22. VGDTININI 23. VLSLAQEQV 24. VTASDPLDT     R     GGSPEK     LGSEGALSP     GGVASLLR 25. YLDKTEQWS     TLPPETK 10         20         30         40 MRLLWGLIWA SSFFTLSLQK PRLLLFSPSV VHLGVPLSVG 50         60         70         80 VQLQDVPRGQ VVKGSVFLRN PSRNNVPCSP K⁽¹⁵⁾ VDFTLSSER 90         100        110        120 DFALLSLQVP LKDAK⁽¹⁸⁾ SCGLH QLLR ⁽¹⁰⁾ GPEVQL VAHSPWLKDS 130        140        150        160 LSRTTNIQGI NLLFSSRR⁽⁷⁾ GH LFLQTDQPIY NPGQRVRYR⁽²¹⁾ V 170        180        190        200 FALDQKMRPS TDTITVMVEN SHGLRVRKKE VYMPSSIFQD 210        220        230        240 DFVIPDISEP GTWKISARFS DGLESNSSTQ FEVKKYVLPN 250        260        270        280 FEVKITPGKP YILTVPGHLD EMQLDIQARY IYGKPVQGVA 290        300        310        320 YVR⁽⁶⁾ FGLLDED GKKTFFRGLE SQTKLVNGQS HISLSK(2)AEFQ 330        340        350        360 DALEK ⁽¹⁴⁾ LNMGI TDLQGLRLYV AAAIIESPGG EMEEAELTSW 370        380        390        400 YFVSSPFSLD LSKTKR⁽¹²⁾ HLVP GAPFLLQALV R ⁽⁴⁾ EMSGSPASG 410        420        430        440 IPVKVSATVS SPGSVPEVQD IQQNTDGSGQ VSIPIIIPQT 450        460        470        480 ISELQLSVSA GSPHPAIARL TVAAPPSGGP GFLSIERPDS 490        500        510        520 RPPR⁽²²⁾ VGDTLN LNLRAVGSGA TFSHYYMIL SRGQIVFMNR 530        540        550        560 EPKRTLTSVS VFVDHHLAPS FYFVAFYYHG DHPVANSLR⁽²⁰⁾ V 570        580        590        600 DVQAGACEGK LELSVDGAKQ YRNGESVKLH LETDSLALVA 610        620        630        640 LGALDTALYA AGSKSHKPLN MGKVFEAMNS YDLGCGPGGG 650        660        670        680 DSALQVFQAA GLAFSDGDQW TLSRKRLSCP KEKTTRKKR⁽¹⁶⁾ N 690        700        710        720 VNFQKAINEK LGQYASPTAK RCCQDGVTRL PMMRSCEQRA 730        740        750        760 ARVQQPDCRE PFLSCCQFAE SLRKKSR⁽³⁾ DKG QAGLQRALEI 770        780        790        800 LQEEDLIDED DIPVRSFFFE NWLWRVETVD RFQILTLWLP 810        820        830        840 DSLTTWEIHG LSLSKTK⁽⁸⁾ GLC VATPVQLRVF REFHLHLRLP 850        860        870        880 MSVRRFEQLE LRPVLYNYLD KNLTVSVHVS PVEGLCLAGG 890        900        910        920 GGLAQQVLVP AGSARPVAFS VVPTAAAAVS LKVVAR⁽¹¹⁾ GSFE 930        940        950        960 FPVGDAVSKV LQIEKEGAIH REELVYELNP LDHRGRTLEI 970        980        990        1000 PGNSDPNMIP DGDFNSYVR⁽²⁴⁾ V TASDPLDTLG SEGALSPGGV 1010       1020       1030       1040 ASLLRLPRGC GEQTMIYLAP TLAASR⁽²⁵⁾ YLDK TEQWSTLPPE 1050       1060       1070       1080 TK ⁽²⁾ DHAVDLIQ KGYMRIQQFR KADGSYAAWL SRDSSTWLTA 1090       1100       1110       1120 FVLK⁽²³⁾ VLSLAQ EQVGGSPEKL QETSNWLLSQ QQADGSFQDP 1130       1140       1150       1160 CPVLDRSMQG GLVGNDETVA LTAFVTIALH HGLAVFQDEG 1170       1180       1190       1200 AEPLKQRVEA SISKANSFLG EKASAGLLGA HAAAITAYAL 1210       1220       1230       1240 TLTKAPVDLL GVAHNNLMAM AQETGDNLYW GSVTGSQSNA 1250       1260       1270       1280 VSPTPAPRNP SDPMPQAPAL WIETTAYALL HLLLHEGKAE 1290       1300       1310       1320 MADQASAWLT R⁽¹⁷⁾ QGSFQGGFR STQDTVIALD ALSAYWIASH 1330       1340       1350       1360 TTEERGLNVT LSSTGRNGFK SHALQLNNRQ IRGLEEELQF 1370       1380       1390       1400 SLGSKINVKV GGNSKGTLKV LRTYNVLDMK NTTCQDLQIE 1410       1420       1430       1440 VTVKGHVEYT MEANEDYEDY EYDELPAKDD PDAPLQPVTP 1450       1460       1470       1480 LQLFEGRRNR RRREAPKVVE EQESRVHYTV CIWRNGKVGL 1490       1500       1510       1520 SGMAIADVTL LSGFHALRAD LEKLTSLSDR YVSHFETEGP 1530       1540       1550       1560 HVLLYFDSVP TSRECVGFEA VQEVPVGLVQ PASATLYDYY 1570       1580       1590       1600 NPERRCSVFY GAPSKSR⁽¹³⁾ LLA TLCSAEVCQC AEGKCPRQRR 1610       1620       1630       1640 ALER⁽⁹⁾ GLQDED GYRMK⁽⁵⁾ FACYY PRVEYGFQVK VLREDSRAAF 1650       1660       1670       1680 RLFETK⁽¹⁸⁾ ITQV LHFTKDVKAA ANQMRNFLVR ASCRLRLEPG 1690       1700       1710       1720 KEYLIMGLDG ATYDLEGHPQ YLLDSNSWIE EMPSERLCRS 1730       1740 TRQRAACAQL NDFLQEYGTQ GCQV Protein Name Uniprot IPI Gene Name Histidine Rich Protein P04196 IPI00022371 HRG_Human Trypsin fragments  1. YKEENDDFA  2. ADLFYDVEA     SFR     LDLESPK MKALIAALLL ITLQYSCAVS PTDCSAVEPE AEKALDLINK                       70         80 RRRDGYLFQL LRIADAHLDR VENTTVYYLV LDVQESDCSV 90         100        110        120 LSRKYWNDCE PPDSRRPSEI VIGQCKVIAT RHSHESQDLR 130        140        150        160 VIDFNCTTSS VSSALANTKD SPVLIDFFED TERYRKQANK 170        180        190        200 ALEK⁽¹⁾ YKEEND DFASFRVDRI ERVARVRGGE GTGYFVDFSV 210        220        230       240 RNCPRHHFPR HPNVFGFCR⁽²⁾ A DLFYDVEALD LESPKNLVIN 250        260        270        280 CEVFDPQEHE NINGVPPHLG HPFHWGGHER SSTTKPPFKP  290        300        310        320 HGSRDHHHPH KPHEHGPPPP PDERDHSHGP PLPQGPPPLL 330        340        350        360 PMSCSSCQHA TFGTNGAQRH SHNNNSSDLH PHKHHSHEQH 370        380        390        400 PHGHHPHAHH PHEHDTHRQH PHGHHPHGHH PHGHHPHGHH 410        420        430        440 PHGHHPHCHD FQDYGPCDPP PHNQGHCCHG HGPPPGHLRR 450        460        470        480 RGPGKGPRPF HCRQIGSVYR LPPLRKGEVL PLPEANPPSF 490        500        510        520 PLPHHKHPLK PDNQPFPQSV SESCPGKFKS GFPQVSMFFT HTFPK Protein Name Uniprot IPI Gene Name C-type lectin domain P05452 IPI00009028.2 CLEC3B_Human family 3, member B (Tetranectin) Trypsin fragments  1. EQQALQTVC  2. TFHEASEDC     LK     ISR 10         20         30         40 MELWGAYLLL CLFSLLTQVT TEPPTQKPKK IVNAKKDVVN 50         60         70         80 TKMFEELKSR LDTLAQEVAL L¹ KEQQALQTV CLKGTKVHMK 90         100        110        120 CFLALTQTK² T FHEASEDCIS RGGTLGTPQT GSENDALYEY 130        140        150        160 LRQSVGNEAE IWLGLNDMAA EGTWVDMTGA RIAYKNWETE 170        180        190        200 ITAQPDGGKT ENCAVLSGAA NGKWFDKRCR DQLPYICQFG IV Protein Name Uniprot IPI Gene Name Kallikrein-8 O60259-2 IPI00219892 KLK8_Human isoform 2 Trypsin fragments  1. ENFPDTLNC     AEVK 10         20         30         40 MGRPRPRAAK TWMFLLLLGG AWAGHSRAQE DKVLGGHECQ 50         60         70         80 PHSQPWQAAL FQGQQLLCGG VLVGGNWVLT AAHCKKPKYT 90         100        110        120 VRLGDHSLQN KDGPEQEIPV VQSIPHPCYN SSDVEDHNHD 130        140        150        160 LMLLQLRDQA SLGSKVKPIS LADHCTQPGQ KCTVSGWGTV 170        180        190        200 TSPR⁽³⁾ ENFPDT LNCAEVKIFP QKKCEDAYPG QITDGMVCAG 210        220        230        240 SSKGADTCQG DSGGPLVCDG ALQGITSWGS DPCGRSDKPG 250        260 VYTNICRYLD WIKKIIGSKG Protein Name Uniprot IPI Gene Name Complement Component P07357 IPI00011252 C8A_Human 8 alpha Trypsin fragments  1. AIDEDCSQY  2. LGSLGAACE  3. QAQCGQDFQ     EPIPGSQK     QTQTEGAK     CK 10         20         30         40 MFAVVFFILS LMTCQPGVTA QEKVNQRVRR AATPAAVTCQ 50         60         70         80 LSNWSEWTDC FPCQDKKYRH RSLLQPNKFG GTICSGDIWD 90         100        110        120 QASCSSSTTC VR⁽³⁾ QAQCGQDF QCKETGRCLK RHLVCNGDQD 130        140        150        160 CLDGSDEDDC EDVR⁽¹⁾ AIDEDC SQYEPIPGSQ KAALGYNILT 170        180        190        200 QEDAQSVYDA SYYGGQCETV YNGEWRELRY DSTCERLYYG 210        220        230        240 DDEKYFRKPY NFLKYHFEAL ADTGISSEFY DNANDLLSKV 250        260        270        280 KKDKSDSFGV TIGIGPAGSP LLVGVGVSHS QDTSFLNELN 290        300        310        320 WYNEKKFIFT RIFTKVQTAH FKMRKDDIML DEGMLQSLME 330        340        350        360 LPDQYNYGMY AKFINDYGTH YITSGSMGGI YEYILVIDKA 370        380        390        400 KMESLGITSR DITTCFGGSL GIQYEDKINV GGGLSGDHCK 410        420        430        440 KFGGGKTERA RKAMAVEDII SRVRGGSSGW SGGLAQNRST 450        460        470        480 ITYRSWGRSL KYNPVVIDFE MQPIHEVLRH TSLGPLEAKR 490        500        510        520 QNLRRALDQY LMEFNACRCG PCFNNGVPIL EGTSCRCQCR 530        540        550        560 ⁽²⁾ LGSLGAACEQ TQTEGAKADG SWSCWSSWSV CRAGIQERRR 570        580 ECDNPAPQNG GASCPGRKVQ TQAC Protein Name Uniprot IPI Gene Name Kallikrein-13 Q9UKR3 IPI00007726 KLK13_Human Trypsin fragments  1. TLQCANIQL  2. ITDNMLCAG     R     TK 10         20         30         40 MWPLALVIAS LTLALSGGVS QESSKVLNTN GTSGFLPGGY 50         60         70         80 TCFPHSQPWQ AALLVQGRLL CGGVLVHPKW VLTAAHCLKE 90         100        110        120 GLKVYLGKHA LGRVEAGEQV REVVHSIPHP EYRRSPTHLM 130        140        150        160 HDHDIMLLEL QSPVQLTGYI QTLPLSHNNR LTPGTTCRVS 170        180        190        200 GWGTTTSPQV NYPR⁽¹⁾ TLQCAN IQLRSDEECR QVYPGK⁽²⁾ ITDN 210        220        230        240 MLCAGTKEGG KDSCEGDSGG PLVCNRTLYG IVSWGDFPCG 250        260        270 QPDRPGVYTR VSRYVLWIRE TIRKYETQQQ KWLKGPQ Protein Name Uniprot IPI Gene Name Complement P10643 IPI00296608 C7_Human Component 7 Trypsin fragments  1. AASGTQNNV  2. DSCTLPASA     LR     EK 10         20         30         40 MKVISLFILV GFIGEFQSFS SASSPVNCQW DFYAPWSECN 50         60         70         80 GCTKTQTRRR SVAVYGQYGG QPCVGNAFET QSCEPTRGCP 90         100        110        120 TEEGCGERFR CFSGQCISKS LVCNGDSDCD EDSADEDRCE 130        140        150        160 DSERRPSCDI DKPPPNIELT GNGYNELTGQ FRNRVINTKS 170        180        190        200 FGGQCRKVFS GDGKDFYRLS GNVLSYTFQV KINNDFNYEF 210        220        230        240 YNSTWSYVKH TSTEHTSSSR KRSFFRSSSS SSRSYTSHTN 250        260        270        280 EIHKGKSYQL LVVENTVEVA QFINNNPEFL QLAEPFWKEL 290        300        310        320 SHLPSLYDYS AYRRLIDQYG THYLQSGSLG GEYRVLFYVD 330        340        350        360 SEKLKENDFN SVEEKKCKSS GWHFVVKFSS HGCKELENAL 370        380        390        400 K⁽¹⁾ AASGTQNNV LRGEPFIRGG GAGFISGLSY LELDNPAGNK 410        420        430        440 RRYSAWAESV TNLPQVIKQK LTPLYELVKE VPCASVKKLY 450        460        470        480 LKWALEEYLD EFDPCHCRPC QNGGLATVEG THCLCHCKPY 490        500        510        520 TFGAACEQGV LVGNQAGGVD GGWSCWSSWS PCVQGKKTRS 530        540        550        560 RECNNPPPSG GGRSCVGETT ESTQCEDEEL EHLRLLEPHC 570        580        590        600 FPLSLVPTEF CPSPPALKDG FVQDEGTMFP VGKNVVYTCN 610        620        630        640 EGYSLIGNPV ARCGEDLRWL VGEMHCQKIA CVLPVLMDGI 650        660        670        680 QSHPQKPFYT VGEKVTVSCS GGMSLEGPSA FLCGSSLKWS 690        700        710        720 PEMKNARCVQ KENPLTQAVP KCQRWEKLQN SRCVCKMPYE 730        740        750        760 CGPSLDVCAQ DERSKRILPL TVCKMHVLHC QGRNYTLTGR 770        780        790        800 ⁽²⁾ DSCTLPASAE KACGACPLWG KCDAESSKCV CREASECEEE 810        820        830        840 GFSICVEVNG KEQTMSECEA GALRCRGQSI SVTSIRPCAA ETQ Protein Name Uniprot IPI Gene Name Retinal P00352 IPI00218914 ALDH1A1_Human Dehydrogenase Trypsin fragments  1. SSSGTPDLP  2. YILGNPLTP     VLLTDLK     GVTQGPQID     KEQYDK 10         20         30         40 M⁽¹⁾ SSSGTPDLP VLLTDLKIQY TKIFINNEWH DSVSGKKFPV 50         60         70         80 FNPATEEELC QVEEGDKEDV DKAVKAARQA FQIGSPWRTM 90         100        110        120 DASERGRLLY KLADLIERDR LLLATMESMN GGKLYSNAYL 130        140        150        160 NDLAGCIKTL RYCAGWADKI QGRTIPIDGN FFTYTRHEPI 170        180        190        200 GVCGQIIPWN FPLVMLIWKI GPALSCGNTV VVKPAEQTPL 210        220        230        240 TALHVASLIK EAGFPPGVVN IVPGYGPTAG AAISSHMDID 250        260        270        280 KVAFTGSTEV GKLIKEAAGK SNLKRVTLEL GGKSPCIVLA 290        300        310        320 DADLDNAVEF AHHGVFYHQG QCCIAASRIF VEESIYDEFV 330        340        350        360 RRSVERAKK⁽²⁾ Y ILGNPLTPGV TQGPQIDKEQ YDKILDLIES 370        380        390        400 GKKEGAKLEC GGGPWGNKGY FVQPTVFSNV TDEMRIAKEE 410        420        430        440 IFGPVQQIMK FKSLDDVIKR ANNTFYGLSA GVFTKDIDKA 450        460        470        480 ITISSALQAG TVWVNCYGVV SAQCPFGGFK MSGNGRELGE 490        500 YGFHEYTEVK TVTVKISQKN S Protein Name Uniprot IPI Gene Name ApoLipoprotein L1 Q9UKR3 IPI00I86903 APOL1_Human isoform 2 Trypsin fragments  1. VTEPISAES     GEQVER 10         20         30         40 MEGAALLRVS VLCIWMSALF LGVGVRAEEA GARVQQNVPS 50         60         70         80 GTDTGDPQSK PLGDWAAGTM DPESSIFIED AIKYFKEKVS 90         100        110        120 TQNLLLLLTD NEAWNGFVAA AELPRNEADE LRKALDNLAR 130        140        150        160 QMIMKDKNWH DKGQQYRNWF LKEFPRLKSE LEDNIRRLRA 170        180        190        200 LADGVQKVHK GTTIANVVSG SLSISSGILT LVGMGLAPFT 210        220        230        240 EGGSLVLLEP GMELGITAAL TGITSSTMDY GKKWWTQAQA 250        260        270        280 HDLVIKSLDK LKEVREFLGE NISNFLSLAG NTYQLTRGIG 290        300        310        320 KDIRALRRAR ANLQSVPHAS ASRPRVTEPI SAESGEQVER 330        340        350        360 VNEPSILEMS RGVKLTDVAP VSFFLVLDVV YLVYESKHLH 370        380        390 EGAKSETAEE LKKVAQELEE KLNILNNNYK ILQADQEL Protein Name Uniprot IPI Gene Name Mucin 1 P15941-2 IPI00218163 Muc1_Human isoform 2 Trypsin fragments  1. DISEMFIQL  2. QGGFLGLSN     YK     IK 10         20         30         40 MTPGTQSPFF LLLLLTVLTV VTGSGHASST PGGEKETSAT 50         60         70         80 QRSSVPSSTE KNAVSMTSSV LSSHSPGSGS STTQGQDVTL 90         100        110        120 APATEPASGS AATWGQDVTS VPVTRPALGS TTPPAHDVTS 130        140        150        160 APDNKPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 170        180        190        200 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 210        220        230        240 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 250        260        270        280 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS  290        300        310        320 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 330        340        350        360 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 370        380        390        400 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 410        420        430        440 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 450        460        470        480 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 490        500        510        520 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 530        540        550        560 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 570        580        590        600 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 610        620        630        640 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 650        660        670        680 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 690        700        710        720 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 730        740        750        760 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 770        780        790        800 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 810        820        830        840 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 850        860        870        880 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 890        900        910        920 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 930        940        950        960 APDTRPAPGS TAPPAHGVTS APDTRPAPGS TAPPAHGVTS 970        980        990        1000 ASGSASGSAS TLVHNGTSAR ATTTPASKST PFSIPSHHSD 1010       1020       1030       1040 TPTTLASHST KTDASSTHHS SVPPLTSSNH STSPQLSTGV 1050       1060       1070       1080 SFFFLSFHIS NLQFNSSLED PSTDYYQELQ R⁽¹⁾ DISEMFLQI 1090       1100       1110       1120 YK⁽²⁾ QGGFLGLS NIKFRPGSVV VQLTLAFREG TINVHDVETQ 1130       1140       1150       1160 FNQYKTEAAS RYNLTISDVS VSDVPFPFSA QSGAGVPGWG 1170       1180       1190       1200 IALLVLVCVL VALAIVYLIA LAVCQCRRKN YGQLDIFPAR 1210       1220       1230       1240 DTYHPMSEYP TYHTHGRYVP PSSTDRSPYE KVSAGNGGSS 1250 LSYTNPAVAA TSANL Protein Name Uniprot IPI Gene Name Bleomycin Q13867 IPI00219575 BLMH_Human Hydrolase Trypsin fragments  1. AQHVFQHAV  2. SSSGLNSEK     PQEGKPITN     VAALIQK     QK 10         20         30         40 M⁽²⁾ SSSGLNSEK VAALIQKLNS DPQFVLAQNV GTTHDLLDIC 50         60         70         80 LKRATVQR⁽¹⁾ AQ HVFQHAVPQE GKPITNQKSS GRCWIFSCLN 90         100        110        120 VMRLPFMKKL NIEEFEFSQS YLFFWDKVER CYFFLSAFVD 130        140        150        160 TAQRKEPEDG RLVQFLLMNP ANDGGQWDML VNIVEKYGVI  170        180        190        200 PKKCFPESYT TEATRRMNDI LNHKMRERCI RLRNLVHSGA 210        220        230        240 TKGEISATQD VMMEEIFRVV CICLGNPPET FTWEYRDKDK 250        260        270        280 NYQKIGPITP LEFYREHVKP LFNMEDKICL VNDPRPQHKY 290        300        310        320 NKLYTVEYLS NMVGGRKTLY NNQPIDFLKK MVAASIKDGE 330        340        350        360 AVWFGCDVGK HFNSKLGLSD MNLYDHELVF GVSLKNMNKA 370        380        390        400 ERLTFGESLM THAMTFTAVS EKDDQDGAFT KWRVENSWGE 410        420        430        440 DHGHKGYLCM TDEWFSEYVY EVVVDRKHVP EEVLAVLEQE 450 PIILPAWDPM GALAE Protein Name Uniprot IPI Gene Name Cornifin-B P22528 IPI00304903 SPRRIB_Human Trypsin fragments  1. QPCTPPPQL  2. VPEPCPSIV     QQQQVK     TPAPAQQK 10         20         30         40 MSSQQQK⁽¹⁾ QPC TPPPQLQQQQ VKQPCQPPPQ EPCIPKTKEP 50         60         70         80 CHPKVPEPCH PKVPEPCQPK VPEPCHPK⁽²⁾ VP EPCPSIVTPA PAQQKTKQK Protein Name Uniprot IPI Gene Name Plasminogen activator P05120 IPI00007117 SERPINB2_Human inhibitor-2 Trypsin fragments  1. GKIPNLLPE     GSVDGDTR 10         20         30         40 MEDLCVANTL FALNLFKHLA KASPTQNLFL SPWSISSTMA 50         60         70         80 MVYMGSRGST EDQMAKVLQF NEVGANAVTP MTPENFTSCG 90         100        110        120 FMQQIQKGSY PDAILQAQAA DKIHSSFRSL SSAINASTGN 130        140        150        160 YLLESVNKLF GEKSASFREE YIRLCQKYYS SEPQAVDFLE 170        180        190        200 CAEEARKKIN SWVKTQTKGK IPNLLPEGSV DGDTRMVLVN 210        220        230        240 AVYFKGKWKT PFEKKLNGLY PFRVNSAQRT PVQMMYLREK 250        260        270        280 LNIGYIEDLK AQILELPYAG DVSMFLLLPD EIADVSTGLE 290        300        310        320 LLESEITYDK LNKWTSKDKM AEDEVEVYIP QFKLEEHYEL 330        340        350        360 RSILRSMGME DAFNKGRANF SGMSERNDLF LSEVFHQAMV 370        380        390        400 DVNEEGTEAA AGTGGVMTGR TGHGGPQFVA DHPFLFLIMA 410 KITNCILFFG RFSSP Protein Name Uniprot IPI Gene Name Peroxiredoxin-6 P30041 IPI00220301 PRDX6_Human Trypsin fragments  1. DINAYNCEE  2. NFDEILR     PTEK 10         20         30         40 MPGGLLLGDV APNFEANTTV GRIRFHDFLG DSWGILFSHP 50         60         70         80 RDFTPVCTTE LGRAAKLAPE FAKRNVKLIA LSIDSVEDHL 90         100        110        120 AWSK⁽¹⁾ DINAYN CEEPTEKLPF PIIDDRNREL AILLGMLDPA 130        140        150        160 EKDEKGMPVT ARVVFVFGPD KKLKLSILYP ATTGR⁽²⁾ NFDEI 170        180        190        200 LRVVISLQLT AEKRVATPVD WKDGDSVMVL PTIPEEEAKK  210        220 LFPKGVFTKE LPSGKKYLRY TPQP Protein Name Uniprot IPI Gene Name Complement Q03591 IPI00011264 CFHR1_Human factor-H Trypsin Fragments  1. ITCTEEGWS  2. STDTSCVNP  3. TGESAEFVC     PTPK     PTVQNAHIL     K     SR 10         20         30         40 MWLLVSVILI SRISSVGGEA TFCDFPKINH GILYDEEKYK 50         60         70         80 PFSQVPTGEV FYYSCEYNFV SPSKSFWTR⁽¹⁾ I TCTEEGWSPT 90         100        110        120 PKCLRLCFFP FVENGRSESS GQTHLEGDTV QIICNTGYRL 130        140        150        160 QNNENNISCV ERGWSTPPKC R⁽²⁾ STDTSCVNP PTVQNAHILS 170        180        190        200 RQMSKYPSGE RVRYECRSPY EMFGDEEVMC LNGNWTEPPQ 210        220        230        240 CKDSTGKCGP PPPIDNGDIT SFPLSVYAPA SSVEYQCQNL 250        260        270        280 YQLEGNKRIT CRNGQWSEPP KCLHPCVISR EIMENYNIAL 290        300        310        320 RWTAKQKLYL R⁽³⁾ TGESAEFVC KRGYRLSSRS HTLRTTCWDG 330 KLEYPTCAKR Protein Name Uniprot IPI Gene Name Isoform 1 of P04217 IPI00022895 A1BG_Human Alpha-1B- glycoprotein Trypsin Fragments  1. ATWSGAVLA  2. CLAPLEGAR  3. GVTFLLR     GR  4. HQFLLTGDT  5. LLELTGPK  6. SGLSTGWTQ     QGR     LSK 10         20         30         40 MSMLVVFLLL WGVTWGPVTE AAIFYETQPS LWAESESLLK 50         60         70         80 PLANVTLTCQ AHLETPDFQL FKNGVAQEPV HLDSPAIK⁽⁴⁾ HQ 90         100        110        120 FLLTGDTQGR YRCR⁽⁸⁾ SGLSTG WTQLSK ⁽⁵⁾ LLEL TGPKSLPAPW 130        140        150        160 LSMAPVSWIT PGLKTTAVCR GVLR⁽⁴⁾ GVTFLL RREGDHEFLE 170        180        190        200 VPEAQEDVEA TFPVHQPGNY SCSYRTDGEG ALSEPSATVT 210        220        230        240 IEELAAPPPP VLMHHGESSQ VLHPGNKVTL TCVAPLSGVD 250        260        270        280 FQLRRGEKEL LVPRSSTSPD RIFFHLNAVA LGDGGHYTCR 290        300        310        320 YRLHDNQNGW SGDSAPVELI LSDETLPAPE FSPEPESGRA 330        340        350        360 LRLR⁽²⁾ CLAPLE GARFALVRED RGGRRVHRFQ SPAGTEALFE 370        380        390        400 LHNISVADSA NYSCVYVDLK PPFGGSAPSE RLELHVDGPP 410        420        430        440 PRPQLR⁽¹⁾ ATWS GAVLAGRDAV LRCEGPIPDV TFELLREGET 450        460        470        480 KAVKTVRTPG AAANLELIFV GPQHAGNYRC RYRSWVPHTF 490 ESELSDPVEL LVAES Protein Name Uniprot IPI Gene Name Gamma-glutamyl Q92820 IPI00023728 GGH_Human hydrolase Trypsin Fragments  1. NLDGISHAP     NAVK 10         20         30         40 MASPGCLLCV LGLLLCGAAS LELSRPHGDT AKKPIIGILM 50         60         70         80 QKCRNKVMKN YGRYYIAASY VKYLESAGAR VVPVRLDLTE 90         100        110        120 KDYEILFKSI NGILFPGGSV DLRRSDYAKV AKIFYNLSIQ 130        140        150        160 SFDDGDYDPV WGTCLGFEEL SLLISGECLL TATDTVDVAM 170        180        190        200 PLNFTGGQLH SRMFQNFPTE LLLSLAVEPL TANFHKWSLS 210        220        230        240 VKNFTMNEKL KKFFNVLTTN TDGKIEFIST MEGYKYPVYG 250        260        270        280 VQWHPEKAPY EWKNLDGISH APNAVKTAFY LAEFFVNEAR 290        300        310 KNNHHFKSES EEERALIYQF SPIYTGHISS FQQCYIFD Protein Name Uniprot IPI Gene Name Ezrin P15311 IPI00843975 EZR_Human Trypsin Fragments  1. ALQLEEER  2. APDFVFYAP  3. ELSEQIQR     R  4. IALLEEAR  5. IGFPWSEIR  6. QRIDEFEAL  7. SGYLSSER  8. SQEQLAAEL  9. VSAQEVRK     AEYTAK 10         20         30         40 MPKPINVRVT TMDAELEFAI QPNTTGKQLF DQVVKTIGLR 50         60         70         80 EVWYFGLHYV DNKGFPTWLK LDKK⁽³⁾ VSAQEV RKENPLQFKF 90         100        110        120 RAKFYPEDVA EELIQDITQK LFFLQVKEGI LSDEIYCPPE 130        140        150        160 TAVLLGSYAV QAKFGDYNKE VHK⁽⁷⁾ SGYLSSE RLIPQRVMDQ 170        180        190        200 HKLTRDQWED RIQVWHAEHR GMLKDNAMLE YLKIAQDLEM 210        220        230        240 YGINYFEIKN KKGTDLWLGV DALGLNIYEK DDKLTPK⁽⁵⁾ IGF 250        260        270        280 PWSEIRNISF NDKKFVIKPI DKK⁽²⁾ APDFVFY APRLRINKRI 290        300        310        320 LQLCMGNHEL YMRRRKPDTI EVQQMKAQAR EEKHQKQLER 330        340        350        360 QQLETEKKRR ETVEREKEQM MREKEELMLR LQDYEEKTKK 370        380        390        400 AER⁽³⁾ ELSEQIQ R ⁽¹⁾ ALQLEEERK RAQEEAERLE ADRMAALRAK 410        420        430        440 EELERQAVDQ IK⁽¹¹⁾ SQEQLAAE LAEYTAK ⁽⁴⁾ IAL LEEARRRKED 450        460        470        480 EVEEWQHRAK EAQDDLVKTK EELHLVMTAP PPPPPPVYEP 490        500        510        520 VSYHVQESLQ DEGAEPTGYS AELSSEGIRD DRNEEKRITE 530        540        550        560 AEKNERVQRQ LLTLSSELSQ ARDENKRTHN DIIHNENMRQ 570        580 GRDKYKTLRQ IRQGNTK⁽⁶⁾ QRI DEFEAL Protein Name Uniprot IPI Gene Name Alpha-2- P08697 IPI00879231 SERPINF2_Human antiplasmin Trypsin Fragments  1. LGNQEPGGQ     TALK 10         20         30         40 MALLWGLLVL SWSCLQGPCS VFSPVSAMEP LGRQLTSGPN 50         60         70         80 QEQVSPLTLL KLGNQEPGGQ TALKSPPGVC SRDPTPEQTH 90         100        110        120 RLARAMMAFT ADLFSLVAQT STCPNLILSP LSVALALSHL 130        140        150        160 ALGAQNHTLQ RLQQVLHAGS GPCLPHLLSR LCQDLGPGAF 170        180        190        200 RLAARMYLQK GFPIKEDFLE QSEQLFGAKP VSLTGKQEDD 210        220        230        240 LANINQWVKE ATEGKIQEFL SGLPEDTVLL LLNAIHFQGF 250        260        270        280 WRNKFDPSLT QRDSFHLDEQ FTVPVEMMQA RTYPLRWFLL 290        300        310        320 EQPEIQVAHF PFKNNMSFVV LVPTHFEWNV SQVLANLSWD 330        340        350        360 TLHPPLVWER PTKVRLPKLY LKHQMDLVAT LSQLGLQELF 370        380        390        400 QAPDLRGISE QSLVVSGVQH QSTLELSEVG VEAAAATSIA 410        420        430        440 MSRMSLSSFS VNRPFLFFIF EDTTGLPLFV GSVRNPNPSA 450        460        470        480 PRELKEQQDS PGNKDFLQSL KGFPRGDKLF GPDLKLVPPM 490 EEDYPQFGSP K Protein Name Uniprot IPI Gene Name Hemopexin P02790 IPI00022488 HPX_Human Trypsin Fragments  1. DVRDYFMPC  2. DYFMPCPGR  3. EVGTPHGII     PGR     LDSVDAAFI     CPGSSR  4. GECQAEGVL  5. GEFVWK  6. GGYTLVSGY     FFQGDR     PK  7. LLQDEFPGI  8. NFPSPVDAA  9. QGHNSVFLI     PSPIDAAVE     FR     K     CHR 10. SGAQATWTE 11. VDGALCMEK 12. WKNFPSPVD     LPWPHEK     AAFR 10         20         30         40 MARVLGAPVA LGLWSLCWSL AIATPLPPTS AHGNVAEGET 50         60         70         80 KPDPDVTERC SDGWSFDATT LDDNGTMLFF K⁽⁵⁾ GEFVWKSHK 90         100        110        120 WDRELISERW K ⁽⁸⁾ NFPSPVDAA FR ⁽⁹⁾ QGHNSVFL IKGDKVWVYP 130        140        150        160 PEKKEKGYPK ⁽⁷⁾ LLQDEFPGIP SPLDAAVECH R ⁽⁴⁾ GECQAEGVL 170        180        190        200 FFQGDREWFW DLATGTMKER SWPAVGNCSS ALRWLGRYYC 210        220        230        240 FQGNQFLRFD PVRGEVPPRY PR⁽¹⁾ DVR ⁽²⁾ DYFMP CPGRGHGHRN 250        260        270        280 GTGHGNSTHH GPEYMRCSPH LVLSALTSDN HGATYAFSGT 290        300        310        320 HYWRLDTSRD GWHSWPIAHQ WPQGPSAVDA AFSWEEKLYL 330        340        350        360 VQGTQVYVFL TK⁽⁶⁾ GGYTLVSG YPKRLEK⁽³⁾ EVG TPHGIILDSV 370        380        390        400 DAAFICPGSS RLHIMAGRRL WWLDLK⁽¹⁰⁾ SGAQ ATWTELPWPH 410        420        430        440 EK ⁽¹¹⁾ VDGALCME KSLGPNSCSA NGPGLYLIHG PNLYCYSDVE 450        460 KLNAAKALPQ PQNVTSLLGC TH Protein Name Uniprot IPI Gene Name Cysteine Rich P54108 IPI00974055 Crisp3_Human Secretory Protein 3 Trypsin Fragments  1. WANQCNYR 10         20         30         40 MTLFPVLLFL VAGLLPSFPA NEDKDPAFTA LLTTQTQVQR 50         60         70         80 EIVNKHNELR RAVSPPARNM LKMEWNKEAA ANAQKWANQC 90         100        110        120 NYRHSNPKDR MTSLKCGENL YMSSASSSWS QAIQSWFDEY 130        140        150        160 NDFDFGVGPK TPNAVVGHYT QVVWYSSYLV GCGNAYCPNQ 170        180        190        200 KVLKYYYVCQ YCPAGNWANR LYVPYEQGAP CASCPDNCDD 210        220        230        240 GLCTNGCKYE DLYSNCKSLK LTLTCKHQLV RDSCKASCNC Protein Name Uniprot IPI Gene Name Carboxypeptidase A4 Q9UI42 IPI00008894 CP4A_Human Trypsin Fragments  1. DPAITSILE  2. SRNPGSSCI  3. GASDNPCSE     K     GADPNR     VYHGPHANS     EVEVK  4. SVVDFIQK  5. NPGSSCIGA     DPNR 10         20         30         40 MRWILFIGAL IGSSICGQEK FFGDQVLRIN VRNGDEISKL 50         60         70         80 SQLVNSNNLK LNFWKSPSSF NRPVDVLVPS VSLQAFKSFL 90         100        110        120 RSQGLEYAVT IEDLQALLDN EDDEMQHNEG QERSSNNFNY 130        140        150        160 GAYHSLEAIY HEMDNIAADF PDLARRVKIG HSFENRPMYV 170        180        190        200 LKFSTGKGVR RPAVWLNAGI HSREWISQAT AIWTARKIVS 210        220        230        240 DYQR⁽¹⁾ DPAITS ILEKMDIFLL PVANPDGYVY TQTQNRLWRK 250        260        270        280 TR⁽²⁾SR⁽⁵⁾ NPGSSC IGADPNRNWN ASFAGK⁽³⁾ GASD NPCSEVYHGP 290        300        310        320 HANSEVEVK ⁽⁴⁾ SVVDFIQKHGN FKGFIDLHSY SQLLMYPYGY 330        340        350        360 SVKKAPDAEE LDKVARLAAK ALASVSGTEY QVGPTCTTVY 370        380        390        400 PASGSSIDWA YDNGIKFAFT FELRDTGTYG FLLPANQIIP 410        420 TAEETWLGLK TIMEHVRDNL Protein Name Uniprot IPI Gene Name N-acetylmuramyl- Q96PD5-2 IPI00394992 PGLYRP2_Human L-alanine amidase Trypsin Fragments  1. GSQTQSHPD  2. TFTLLDPK     LGTEGCWDQ     LSAPR 10         20         30         40 MAQGVLWILL GLLLWSDPGT ASLPLLMDSV IQALAELEQK 50         60         70         80 VPAAKTRHTA SAWLMSAPNS GPHNRLYHFL LGAWSLNATE 90         100        110        120 LDPCPLSPEL LGLTKEVARH DVREGKEYGV VLAPDGSTVA 130        140        150        160 VEPLLAGLEA GLQGRRVINL PLDSMAAPWE TGDTFPDVVA 170        180        190        200 IAPDVRATSS PGLRDGSPDV TTADIGANTP DATKGCPDVQ 210        220        230        240 ASLPDAKAKS PPTMVDSLLA VTLAGNLGLT FLR⁽¹⁾ GSQTQSH 250        260        270        280 PDLGTEGCWD QLSAPR ⁽²⁾ TFTL LDPKASLLTM AFLNGALDGV 290        300        310        320 ILGDYLSRTP EPRPSLSHLL SQYYGAGVAR DPGFRSNFRR 330        340        350        360 QNGAALTSAS ILAQQVWGTL VLLQRLEPVH LQLQCMSQEQ 370        380        390        400 LAQVAANATK EFTEAFLGCP ATHPRCRWGA APYRGRPKLL 410        420        430        440 QLPLGFLYVH HTYVPAPPCT DFTRCAANMR SMQRYHQDTQ 450        460        470        480 GWGDIGYSFV VGSDGYVYEG RGWHWVGAHT LGHNSRGFGV 490        500        510        520 AIVGNYTAAL PTEAALRTVR DTLPSCAVRA GLLRPDYALL 530        540        550        560 GHRQLVRTDC PGDALFDLLR TWPHFTATVK PRPARSVSKR 570 SRREPPPRTL PATDLQ Protein Name Uniprot Gene Name Caspase-14 P31944 CASP14_Human Trypsin Fragments  1. AREGSEEDL  2. DPTAEQFQE  3. FQQAIDSR     DALEHMFR     ELEK  4. KTNPEIQST  5. MAEAELVQE  6. RDPTAEQFQ     LR     GK     EELEK  7. RMAEAELVQ  8. SLEEEKYDM  9. TNPEIQSTL     EGK     SGAR     R 10. VYIIQACR 10         20         30         40 MSNPR⁽⁸⁾ SLEEE KYDMSGARLA LILCVTK⁽¹⁾ ARE GSEEDLDALE  50         60         70         80 HMFRQLRFES TMK⁽⁶⁾ R ⁽²⁾ DPTAEQ   FQEELEK ⁽³⁾ FQQ AIDSREDPVS 90         100        110        120 CAFVVLMAHG REGFLKGEDG EMVKLENLFE ALNNKNCQAL 130        140        150        160 RAKPKV⁽¹⁰⁾ YIIQ ACRGEQRDPG ETVGGDEIVM VIKDSPQTIP 170        180        190        200 TYTDALHVYS TVEGYIAYRH DQKGSCFIQT LVDVFTKRKG 210        220        230        240 HILELLTEVT R⁽⁷⁾ R⁽⁵⁾ MAEAELVQ   EGK AR⁽⁴⁾ K ⁽⁹⁾ TNPE  IQSTLR KRLY LQ Protein Name Uniprot IPI Gene Name Ig Kappa chain P04207 IPI00385253 KV308_Human V-III region POM Trypsin Fragments  1. LLIYGASTR 10         20         30         40 MEAPAQLLFL LLLWLPDTTG SIVMTQSPAT LSVSPGERAT 50         60         70         80 LSCRASQSVS NNLAWYQQKP GQPPRLLIYG ASTRATGIPA 90         100        110        120 RFSGSGSGTE FTLTISRLQS EDFAVYYCQQ YNNWPPWTFG QGTRVEIKR Protein Name Uniprot IPI Gene Name Ig Kappa chain P01624 IPI00387119 KV306_Human V-III region POM Trypsin Fragments  1. EIVMTQSPV     TLSVSPGER 10         20         30         40 EIVMTQSPVT LSVSPGERAT LSCRASQSIS NSYLAWYQQK 50         60         70         80 PSGSPRLLIY GASTRATGIP ARFSGSGSGT EFTLTISSLQ 90         100 SEDFAVYYCQ QYNNWPPTFG QGTRVEIKR Protein Name Uniprot IPI Gene Name Isoform 1 P02768-1 IPI00387119 ALB_Human Serum Albumin Trypsin Fragments  1. AACLLPK  2. AAFTECCQA  3. AAFTECCQA     ADK     ADKAACLLP     K  4. ADDKETCFA  5. ADDKETCFA  6. AEFAEVSK     EEGK     EEGKK  7. AEFAEVSKL  8. ATKEQIK  9. ATKEQIKAV     VTDLTK     MDDFAAFVE     K 11. AVMDDFAAF 12. CASIQKFGE 13. CCAAADPHE     VEK     R     CYAK 14. CCKADDKET 15. CCKHPEAK 16. CCTESLVNR     CFAEEGK 17. CCTESLVNR 18. DDNPNLPR 19. DAHKSEVAH     RPCFSALEV     R     DETYVPK 20. DLGEENFK 21. DVCKNYAEA 22. DVFLGMFLY     K     EYAR 23. ECCEKPLLE 24. EFNAETFTF 25. EFNAETFTF     K     HADICTLSE     HADICTLSE     K     KER 26. EQLKAVMDD 27. ETCFAEGK 28. ETCFAEEGK     FAAFVEK     K 29. ETYGEMADC 30. FKDLGEENF 31. FPKAEFAEV     CAK     K     SK 32. FQNALLVR 33. HPDYSVVLL 34. HPYFYAPEL     LR     LFFAK 35. LAKTYETTL 36. LCTVATLR 37. LCTVATLRE     EK     TYGEMADCC     AK 38. LDELRDEGK 39. LDELRDEGK 40. LKCASLQK     ASSAK 41. LKECCEKPL 42. LSQRFPK 43. LFSQRFPKA     LEK     EFAEVSK 44. LVAASQAAL 45. LVNEVTEFA 46. IVNEVTEFA     GL     K     KTCVADESA     ENCDK 47. LVRPEVDVM 48. LVRPEVDVM 49. LVTDLTK     CTAFHDNEE     CTAFHDNEE     TFLK     TFLKK 50. KLVAASQAA 51. KQTALVELV 52. KVPQVSTPT     LGL     K     LLVEVSR 53. KYLYEIAR 54. MPCAEDYIL 55. NECFIQHK     SVVILNQIL     CVILHEK 56. NECFLQHKD 57. NIGKVGSK 58. NYAEAK     DNPNLPR 59. NYAEAKDVF 60. PLVEEPQNL 61. QEPERNECF     IGMFIYEYA     IK     LQHK     R 62. QEPERNECF 63. QNCELFEQL 64. QNCELFEQL     LQHKDDNPN     GEYK     GEYKFQNAI     LPR     IVR 65. QTAIVEIVK 66. RHPDYSVVL 67. RMPCAEDYL     LLR     SVVLKNQLC     VLHEK 68. RPCFSALEV 69. SHCIAEVEN 70. SHCIAEVEN     DETYVPK     DEMPADLPS     DEMPADLPS     LAADFVESK     LAADFVESK     DVCKNYAEA     K 71. SHCIAEVEN 72. SLHTLFGDK 73. SLHTLFGDK     DEMPADLPS     LCTVATLR     LAADFVESK     DVCK 74. TCVADESAE 75. TCVADESAE 76. TCVADESAE     NCDK     NCDKSLHTL     NCDKSLHTL     FGDK     FGDKLCTVA     TLR 77. TPVSDRVTK 78. TYETTLEK 79. TYETTIEKC     CAAADPHEC     YAK 80. VFDEFKPLV 81. VHTECCHGD 82. VHTECCHGD     EEPQNLIK     LLECADDR     LLECADDRA     DLAK 83. VHTECCHGD 84. VPQVSTPTL 85. YICENQDSI     LLECADDRA     VEVSR     SSK     DLAKYICEN     QDSISSK 86. YICENQDSI 87. YLYEIAR 88. YLYEIARR     SSKLK 89. YKAAFTECC 90. YKAAFTECC     QAADK     QAADKAACL     LPK MKWVTFISLL FLFSSAYSRG VFRR⁽¹⁰⁾ DAHKSE VAHR ⁽³⁰⁾ FK ⁽²⁰⁾ DLGE ENFKALVLIA FAQYLQQCPFEDHVK⁽⁴⁵⁾ LVNEV TEFAK ^((74,75,76)) TCVAD ESAENCDK ^((72,73)) SL HTLFGDK ^((37,36)) LCT VATLR ⁽²³⁾ ETYGE MADCCAK ⁽⁶¹⁾⁽⁶²⁾ QEP ER ⁽⁵⁵⁾⁽⁵⁶⁾ NECFLQHK ⁽¹⁸⁾ DDNPNLPR ⁽⁴⁷⁾⁽⁴⁸⁾ LV RPEVDVMCTA FHDNEETFLK  ⁽⁵³⁾ K ^((87,88)) YLYEIARR⁽²⁴⁾ H PYFYAPELLF FAKR^((89,90)) YK ⁽²⁾⁽³⁾ AAFT ECCQAADK ⁽¹⁾ AA CLLPK ⁽³⁹⁾⁽³⁸⁾ LDELR DEGKASSAKQ R⁽⁴⁹⁾ LK ⁽¹²⁾ CASLQKFGERAFKAWAV AR⁽⁴³⁾⁽⁴⁸⁾LSQR⁽³¹⁾ FPK ⁽⁴⁾ A EFAEV ⁽⁷⁾ SK ⁽⁴⁹⁾ LVT DLTK ^((81,82,83)) VHTECC HGDLLECADD RADLAK ^((85,86)) YICE NQDSISSK ⁽⁴¹⁾ LK ⁽²³⁾ ECCEKPLLEK ^((69,70,71)) SHCIAEVEND EMPADLPSLA ADFVESK ⁽²¹⁾ DVC K ⁽⁵⁸⁾⁽⁵⁹⁾ NYAEAK ⁽²²⁾ DVFLGMFLYEYAR ⁽⁶⁸⁾ R ⁽³³⁾ HPDYSVVLL LR ⁽³⁵⁾ LAK ^((78,79)) TYETT LEK ⁽¹²⁾ CCAAADP HECYAK ⁽⁸⁰⁾ VFDE FK ⁽⁴⁰⁾ PLVEEPQN LIK ⁽⁶³⁾⁽⁶⁴⁾ QNCELFE QLGEYK ⁽³²⁾ FQNA LLVRYTK⁽⁵²⁾ K ⁽⁵⁴⁾ VP QVSTPTLVEV SR ⁽⁵⁷⁾ NLGKVGSK ⁽¹⁵⁾ CCKHPEAK ⁽⁶⁷⁾R⁽⁶⁴⁾ M PCAEDYLSVV LNQLCVLHEK ⁽⁷⁷⁾ TPVSDRVTK ⁽¹⁷⁾⁽¹⁶⁾ C CTESLVNR ⁽⁶⁸⁾ RP CFSALEVDET YVPK ⁽²⁵⁾⁽²⁴⁾ EFNAETFTF HADICTL SEKERQIK⁽⁵¹⁾ K ⁽⁶⁵⁾ Q TALVELVKHK PK⁽¹⁰⁾⁽⁹⁾ ATK ⁽²⁶⁾ EQLK ⁽¹¹⁾ A VMDDFAAFVEK ⁽¹⁴⁾ CCK ⁽⁴⁾ ADDK ⁽²⁸⁾⁽²⁷⁾ ET CFAEEGK ⁽⁵⁾⁽⁵⁶⁾ K ⁽⁴⁴⁾ LV AASQAALGL Protein Name Uniprot IPI Gene Name Isoform 1 of P08603 IPI00029739 CFH_Human Complement factor H Trypsin Fragments  1. *AGEQVTYT  2. CLHPCVISR  3. *DGWSAQPT     CATYYK     CIK  4. *DTSCVNPP  5. *EFDHNSNI  6. EIMENYNIA     TVQNAYIVS     R     LR     R  7. GDAVCTESG  8. GDAVCTESG  9. *IDVHLVPD     WR     WRPLPSCEE     R     K 10. *LSYTCEGG 11. IVSSAMEPD 12. NTEILTGSW     FR     REYHFGQAV     SDQTYPEGT     R     QAIYK 13. RPYFPVAVG 14. *SCDIPVFM 15. SIDVACHPG     K     NAR     YALPK 16. SLGNVIMVC 17. *SSNLIILE 18. *SSQESYAH     R     EHLK     GTK 19. TGDEITYQC 20. TGESVEFVC 21. *TKNDFTWF     R     K     K 22. TTCWDGKLE 23. *VSVLCQEN 24. *WQSIPLCV     YPTCAK     YLIQEGEEL     EK     TCKDGR 25. *WSSPPQCE     GLPCK 10         20         30         40 MRLLAKIICL MLWAICVAED CNELPPRR⁽¹²⁾ NT EILTGSWSDQ 50         60         70         80 TYPEGTQAIY KCRPGYR⁽¹⁶⁾ SLG NVIMVCRKGE WVALNPLRKC 90         100        110        120 QKRPCGHPGD TPEGTFTLTG GNVFEYGVKA VYTCNEGYQL 130        140        150        160 LGEINYRECD TDGWTNDIPI CEVVKCLPVT APENGK⁽¹¹⁾ IVSS 170        180        190        200 AMEPDREYHF GQAVRFVCNS GYKIEGDEEM HCSDDGFWSK 210        220        230        240 EKPKCVEISC KSPDVINGSP ISQKIIYKEN ERFQYKCNMG 250        260        270        280 YEYSER⁽⁷⁾ GDAV CTESGWR ⁽⁸⁾ PLP SCEEKSCDNP YIPNGDYSPL 290        300        310        320 RIKHR⁽¹⁹⁾ TGDEI TYQCRNGFYP ATRGNTAKCT STGWIPAPRC  330        340        350        360 TLKPCDYPDI KHGGLYHENM R⁽¹³⁾ RPYFPVAVG KYYSYYCDEH 370        380        390        400 FETPSGSYWD HIHCTQDGWS PAVPCLRKCY FPYLENGYNQ 410        420        430        440 NYGRKFVQGK ⁽¹⁵⁾ SIDVACHPGY ALPKAQTTVT CMENGWSPTP 450        460        470        480 RCIRVKTCSK SSIDIENGFI SESQYTYALK EKAKYQCKLG 490        500        510        520 YVTADGETSG SITCGK⁽³⁾ DGWS AQPTCIK ⁽¹⁴⁾ SCD IPVFMNAR ⁽²¹⁾ TK 530        540        550        560 NDFTWFKLND TLDYECHDGY ESNTGSTTGS IVCGYNGWSD 570        580        590        600 LPICYERECE LPK⁽⁹⁾ IDVHLVP DRKKDQYKVG EVLKFSCKPG 610        620        630        640 FTIVGPNSVQ CYHFGLSPDL PICKEQVQSC GPPPELLNGN 650        660        670        680 VKEKTKEEYG HSEVVEYYCN PRFLMKGPNK IQCVDGEWTT 690        700        710        720 LPVCIVEEST CGDIPELEHG WAQLSSPPYY YGDSVEFNCS 730        740        750        760 ESFTMIGHRS ITCIHGVWTQ LPQCVAIDKL KKCK⁽¹⁷⁾ SSNLII 770        780        790        800 LEEHLKNKK⁽⁵⁾ E FDHNSNIRYR CRGKEGWIHT VCINGRWDPE 810        820        830        840 VNCSMAQIQL CPPPPQIPNS HNMTTTLNYR DGEK⁽²³⁾ VSVLCQ 850        860        870        880 ENYLIQEGEE ITCKDGR ⁽²⁴⁾ WQS IPLCVEKIPC SQPPQIEHGT 890        900        910        920 INSSR⁽¹⁸⁾ SSQES YAHGTK ⁽¹⁰⁾ LSYT CEGGFRISEE NETTCYMGK⁽²⁵⁾ W 930        940        950        960 SSPPQCEGLP CKSPPEISHG VVAHMSDSYQ YGEEVTYKCF 970        980        990        1000 EGFGIDGPAI AKCLGEKWSH PPSCIKTDCL SLPSFENAIP 1010       1020       1030       1040 MGEKKDVYK⁽¹⁾ A GEQVTYTCAT YYKMDGASNV TCINSRWTGR 1050       1060       1070       1080 PTCR⁽⁴⁾ DTSCVN PPTVQNAYIV SRQMSKYPSG ERVRYQCRSP 1090       1100       1110       1120 YEMFGDEEVM CLNGNWTEPP QCKDSTGKCG PPPPIDNGDI 1130       1140       1150       1160 TSFPLSVYAP ASSVEYQCQN LYQLEGNKRI TCRNGQWSEP 1170       1180       1190       1200 PK⁽²⁾ CLHPCVIS R ⁽⁶⁾ EIMENYNIA LRNTAKQKLY SR⁽²⁰⁾ TGESVEFV 1210       1220       1230 CKRGYRLSSR SHTLR⁽²²⁾ TTCWD GKLEYPTCAK R Protein Name Uniprot IPI Gene Name Isoform 1 of Sodium- O95436-1 IPI00007910 SLC34A2_Human dependent phosphate transport protein Trypsin Fragments  1. EAQGEVPAS  2. VISQIAMND     DSKTECTAL     EK 10         20         30         40 MAPWPELGDA QPNPDKYLEG AAGQQPTAPD KGKETNKTDN 50         60         70         80 TEAPVTKIEL LPSYSTATLI DEPTEVDDPW NLPTLQDSGI 90         100        110        120 KWSERDTKGK ILCFFQGIGR LILLLGFLYF FVCSLDILSS 130        140        150        160 AFQLVGGKMA GQFFSNSSIM SNPLLGLVIG VLVTVLVQSS 170        180        190        200 STSTSIVVSM VSSSLLTVRA AIPIIMGANI GTSITNTIVA 210        220        230        240 LMQVGDRSEF RRAFAGATVH DEFNWLSVLC LLPVEVATHY 250        260        270        280 LEIITQLIVE SFHFKNGEDA PDLLKVITKP FTKLIVQLDK 290        300        310        320 K⁽²⁾ VISQIAMND EKAKNKSLVK IWCKTFTNKT QINVTVPSTA 330        340        350        360 NCTSPSLCWT DGIQNWTMKN VTYKENIAKC QHIFVNFHLP 370        380        390        400 DLAVGTILLI LSLLVLCGCL IMIVKILGSV LKGQVALVIK 410        420        430        440 KTINTDFPFP FAWLTGYLAI LVGAGMTFIV QSSSVFTSAL 450        460        470        480 TPLIGIGVIT IERAYPLTLG SNIGTTTTAI LAALASPGNA 490        500        510        520 LRSSLQIALC HFFFNISGIL LWYPIPFTRL PIRMAKGLGN 530        540        550        560 ISAKYRWFAV FYLIIFFFLI PLTVFGLSLA GWRVLVGVGV 570        580        590        600 PVVFIIILVL CLRLLQSRCP RVLPKKLQNW NFLPLWMRSL 610        620        630        640 KPWDAVVSKF TGCFQMRCCC CCRVCCRACC LLCDCPKCCR 650        660        670        680 CSKCCEDLEE AQEGQDVPVK APETFDNITI SR⁽¹⁾ EAQGEVPA 690 SDSKTECTAL Protein Name Uniprot IPI Gene Name Putative IPI00152189 Uncharacterized protein Trypsin Fragments  1. FSVLGSGLN     R MAWAPLLLTLLSLLTGSLSQPVLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRPG KGPRFVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDESDYHCGADHGSGS NFV Protein Name Uniprot IPI Gene Name Ras Related Protein Q15771 IPI00302030 RAB_30_Human Rab-30 Trypsin Fragments  1. LQIWDTAGQ  2. SMEDYDFLF     ER     K 10         20         30         40 M⁽²⁾ SMEDYDFLF KIVLIGNAGV GKTCLVRRFT QGLFPPGQGA 50         60         70         80 TIGVDFMIKT VEINGEKVK⁽²⁾ L QIWDTAGQER FRSITQSYYR 90         100        110        120 SANALILTYD ITCEESFRCL PEWLREIEQY ASNKVITVLV 130        140        150        160 GNKIDLAERR EVSQQRAEEF SEAQDMYYLE TSAKESDNVE 170        180        190        200 KLFLDLACRL ISEAPQNTLV NHVSSPLPGE GKSISYLTCC  NFN

APPENDIX II

Sequences shown to be down regulated in subjects with cancer:

Protein Name Uniprot IPI Gene Name Isoform 1 of growth Q14393 IPI00412410 GAS6_Human arrest-specific protein 6 Trypsin Fragments  1. CEQVCVNSP  2. GQSEVSAAQ  3. IAVAGDLFQ     GSYTCHCDG     LQER     PER     R  4. MFSGTPVIR  5. MQCFSVTER  6. NSGFATCVQ     NLPDQCTPN     PCDR 10         20         30         40 MAPSLSPGPA ALRRAPQLLL LLLAAECALA ALLPAREATQ 50         60         70         80 FLRPRQRRAF QVFEEAKQGH LERECVEELC SREEAREVFE 90         100        110        120 NDPETDYFYP RYLDCINKYG SPYTK⁽⁸⁾ NSGFA TCVQNLPDQC 130        140        150        160 TPNPCDRKGT QACQDLMGNF FCLCKAGWGG RLCDKDVNEC 170        180        190        200 SQENGGCLQI CHNKPGSFHC SCHSGFELSS DGRTCQDIDE 210        220        230        240 CADSEACGEA RCKNLPGSYS CLCDEGFAYS SQEKACRDVD 250        260        270        280 ECLQGR⁽¹⁾ CEQV CVNSPGSYTC HCDGRGGLKL SQDMDTCELE 290        300        310        320 AGWPCPRHRR DGSPAARPGR GAQGSRSEGH IPDRRGPRPW 330        340        350        360 QDILPCVPFS VAKSVKSLYL GR⁽⁴⁾ MFSGTPVI RLRFKRLQPT 370        380        390        400 RLVAEFDFRT FDPEGILLFA GGHQDSTWIV LALRAGRLEL 410        420        430        440 QLRYNGVGRV TSSGPVINHG MWQTISVEEL ARNLVIKVNR 450        460        470        480 DAVMK⁽³⁾ IAVAG DLFQPERGLY HLNLTVGGIP FHEKDLVQPI 490        500        510        520 NPRLDGCMRS WNWLNGEDTT IQETVKVNTR ⁽⁵⁾ MQCFSVTERG 530        540        550        560 SFYPGSGFAF YSLDYMRTPL DVGTESTWEV EVVAHIRPAA 570        580        590        600 DTGVLFALWA PDLRAVPLSV ALVDYHSTKK LKKQLVVLAV 610        620        630        640 EHTALALMEI KVCDGQEHVV TVSLRDGEAT LEVDGTR⁽²⁾ GQS 650        660        670        680 EVSAAQLQER LAVLERHLRS PVLTFAGGLP DVPVTSAPVT 690        700        710        720 AFYRGCMTLE VNRRLLDLDE AAYKHSDITA KSCPPVEPAA Protein Name Uniprot IPI Gene Name Cathepsin L1 P07711 IPI00012887 CTSL1_Human Trypsin Fragments  1. HSFTMAMNA  2. LYGMNEEGW  3. NHCGIASAA     FGDMTSEEF     R     SYPV     R  4. NSWGEEWGM     GGYVK 10         20         30         40 MNPTLILAAF CLGIASATLT FDHSLEAQWT KWKAMGNR⁽²⁾ LY 50         60         70         80 GMNEEGWRRA VWEKNMKMIE LHNQEYREGK ⁽¹⁾ HSFTMAMNAF 90         100        110        120 GDMTSEEFRQ VMNGFQNRKP RKGKVFQEPL FYEAPRSVDW 130        140        150        160 REKGYVTPVK NQGQCGSCWA FSATGALEGQ MFRKTGRLIS 170        180        190        200 LSEQNLVDCS GPQGNEGCNG GLMDYAFQYV QDNGGLDSEE 210        220        230        240 SYPYEATEES CKYNPKYSVA NDTGFVDIPK QEKALMKAVA 250        260        270        280 TVGPISVAID AGHESFLFYK EGIYFEPDCS SEDMDHGVLV 290        300        310        320 VGYGFESTES DNNKYWLVK⁽⁴⁾ N SWGEEWGMGG YVKMAKDRR⁽³⁾ N 330 HCGIASAASY PTV Protein Name Uniprot IPI Gene Name Secreted frizzled- Q8N474 IPI00749245 SFRP1_Human related protein 1 Trypsin Fragments  1. FYTKPPQCV  2. LCHNVGYK  3. LCHNVGYKK     DIPADLR  4. MVLPNLLEH  5. PQGTTVCPP  6. QQASSWVPL     ETMAEVK     CDNELK     LNK  7. SEAIIEHLC  8. SQYLLTAIH     ASEFALR     K 10         20         30         40 MGIGRSEGGR RGAALGVLLA LGAALLAVGS ASEYDYVSFQ 50         60         70         80 SDIGPYQSGR ⁽¹⁾ FYTKPPQCVD IPADLR ^((2,3)) LCHN VGYKK⁽⁴⁾ MVLPN 90         100        110        120 LLEHETMAEV K ⁽⁶⁾ QQASSWVPL LNKNCHAGTQ VFLCSLFAPV 130        140        150        160 CLDRPIYPCR WLCEAVRDSC EPVMQFFGFY WPEMLKCDKF 170        180        190        200 PEGDVCIAMT PPNATEASK⁽⁵⁾ P QGTTVCPPCD NELK ⁽⁷⁾ SEAIIE 210        220        230        240 HLCASEFALR MKIKEVKKEN GDKKIVPKKK KPLKLGPIKK 250        260        270        280 KDLKKLVLYL KNGADCPCHQ LDNLSHHFLI MGRKVK⁽⁸⁾ SQYL 290        300        310 LTAIHKWDKK NKEFKNFMKK MKNHECPTFQ SVFK Protein Name Uniprot IPI Gene Name Bactericidal  P17213 IPI00827847 BPI_Human permeability- increasing protein Trypsin Fragments  1. GLDYASQQG  2. IKIPDYSDS     TAALQK     FK 10         20         30         40 MRENMARGFC NAPRWASLMV LVAIGTAVTA AVNPGVVVRI 50         60         70         80 SQK⁽²⁾ GLDYASQ QGTAALQKEL KR⁽²⁾ IKIPDYSD SFKIKHLGKG 90         100        110        120 HYSFYSMDIR EFQLPSSQIS MVPNVGLKFS ISNANIKISG 130        140        150        160 KWKAQKRFLK MSGNFDLSIE GMSISADLKL GSNPTSGKPT 170        180        190        200 ITCSSCSSHI NSVHVHISKS KVGWLIQLFH KKIESALRNK 210        220        230        240 MNSQVCEKVT NSVSSELQPY FQTLPVMTKI DSVAGINYGL 250        260        270        280 VAPPATTAET LDVQMKGEFY SENHHNPPPF APPVMEFPAA 290        300        310        320 HDRMVYLGLS DYFFNTAGLV YQEAGVLKMT LRDDMIPKES 330        340        350        360 KFRLTTKFFG TFLPEVAKKF PNMKIQIHVS ASTPPHLSVQ 370        380        390        400 PTGLTFYPAV DVQAFAVLPN SSLASLFLIG MHTTGSMEVS 410        420        430        440 AESNRLVGEL KLDRLLLELK HSNIGPFPVE LLQDIMNYIV 450        460        470        480 PILVLPRVNE KLQKGFPLPT PARVQLYNVV LQPHQNFLLF GADVVYK Protein Name Uniprot IPI Gene Name Chitinase domain Q9BWS9-2 IPI00306719 CHID1_Human containing protein 1 Trypsin Fragments  1. GLHIVPR  2. GLVVTDLK  3. NVLDSEDEI     EELSK  4. SQFSDKPVQ  5. YIQTLK     DR 10         20         30         40 MRTLFNLLWL ALACSPVHTT LSKSDAKKAA SKTLLEK⁽⁴⁾ SQF 50         60         70         80 SDKPVQDR ⁽²⁾ GL VVTDLKAESV VLEHRSYCSA KARDRHFAGD 90         100        110        120 VLGYVTPWNS HGYDVTKVFG SKFTQISPVW LQLKRRGREM 130        140        150        160 FEVTGLHDVD QGWMRAVRKH AK⁽¹⁾ GLHIVPRL LFEDWTYDDF 170        180        190        200 R⁽³⁾ NVLDSEDEI EELSKTVVQV AKNQHFDGFV VEVWNQLLSQ 210        220        230        240 KRVGLIHMLT HLAEALHQAR LLALLVIPPA ITPGTDQLGM 250        260        270        280 FTHKEFEQLA PVLDGFSLMT YDYSTAHQPG PNAPLSWVRA 290        300        310        320 CVQVLDPKSK WRSKILLGLN FYGMDYATSK DAREPVVGAR 330        340        350        360 ⁽⁵⁾ YIQTLKDHRP RMVWDSQASE HFFEYKKSRS GRHVVFYPTL 370        380        390 KSLQVRLELA RELGVGVSIW ELGQGLDYFY DLL Protein Name Uniprot IPI Gene Name Moesin P26038 IPI00219365 MSN_Human Trypsin Fragments  1. ALELEQER  2. ALTSELANA  3. AQMVQEDLE     R     K  4. ESEAVEWQQ  5. ISQLEMAR  6. IGFPWSEIR     K 10         20         30         40 MPKTISVRVT TMDAELEFAI QPNTTGKQLF DQVVKTIGLR 50         60         70         80 EVWFFGLQYQ DTKGFSTWLK LNKKVTAQDV RKESPLLFKF 90         100        110        120 RAKFYPEDVS EELIQDITQR LFFLQVKEGI LNDDIYCPPE 130        140        150        160 TAVLLASYAV QSKYGDFNKE VHKSGYLAGD KLLPQRVLEQ 170        180        190        200 HKLNKDQWEE RIQVWHEEHR GMLREDAVLE YLKIAQDLEM 210        220        230        240 YGVNYFSIKN KKGSELWLGV DALGLNIYEQ NDRLTPK⁽⁵⁾ IGF 250        260        270        280 PWSEIRNISF NDKKFVIKPI DKKAPDFVFY APRLRINKRI 290        300        310        320 LALCMGNHEL YMRRRKPDTI EVQQMKAQAR EEKHQKQMER 330        340        350        360 AMLENEKKKR EMAEKEKEKI EREKEELMER LKQIEEQTKK 370        380        390        400 AQQELEEQTR R⁽¹⁾ ALLEQERK RAQSEAEKLA KERQEAEEAK 410        420        430        440 EALLQASRDQ KKTQEQLALE MAELTAR⁽⁵⁾ ISQ LEMARQKK⁽⁴⁾ ES 450        460        470        480 EAVEWQQK ⁽³⁾ AQ MVQEDLEKTR AELKTAMSTP HVAEPAENEQ 490        500        510        520 DEQDENGAEA SADLRADAMA KDRSEEERTT EAEKNERVQK 530        540        550        560 HLK⁽²⁾ ALTSELA NARDESKKTA NDMIHAENMR LGRDKYKTLR 570 QIRQGNTKQR IDEFESM Protein Name Uniprot IPI Gene Name Isoform 2 of Q9NZ08-2 IPI00165949 ERAP1_Human Endoplasmic reticulum aminopeptidase 1 Trypsin Fragments  1. GACILNMLR  2. ILASTQFEP  3. SQIEFALCR     TAAR 10         20         30         40 MVFLPLKWSL ATMSFLLSSL LALLTVSTPS WCQSTEASPK 50         60         70         80 RSDGTPFPWN KIRLPEYVIP VHYDLLIHAN LTTLTFWGTT 90         100        110        120 KVEITASQPT STIILHSHHL QISRATLRKG AGERLSEEPL 130        140        150        160 QVLEHPRQEQ IALLAPEPLL VGLPYTVVIH YAGNLSETFH  170        180        190        200 GFYKSTYRTK EGELR⁽²⁾ ILAST QFEPTAARMA FPCFDEPAFK 210        220        230        240 ASFSIKIRRE PRHLAISNMP LVKSVTVAEG LIEDHFDVTV 250        260        270        280 KMSTYLVAFI ISDFESVSKI TKSGVKVSVY AVPDKINQAD 290        300        310        320 YALDAAVTLL EFYEDYFSIP YPLPKQDLAA IPDFQSGAME 330        340        350        360 NWGLTTYRES ALLFDAEKSS ASSKLGITMT VAHELAHQWF 370        380        390        400 GNLVTMEWWN DLWLNEGFAK FMEFVSVSVT HPELKVGDYP 410        420        430        440 FGKCFDAMEV DALNSSHPVS TPVENPAQIR EMFDDVSYDK 450        460        470        480 ⁽¹⁾ GACILNMLRE YLSADAFKSG IVQYLQKHSY KNTKNEDLWD 490        500        510        520 SMASICPTDG VKGMDGFCSR SQHSSSSSHW HQEGVDVKTM 530        540        550        560 MNTWTLQKGF PLITITVRGR NVHMKQEHYM KGSDGAPDTG 570        580        590        600 YLWHVPLTFI TSKSDMVHRF LLKTKTDVLI LPEEVEWIKF 610        620        630        640 NVGMNGYYIV HYEDDGWDSL TGLLKGTHTA VSSNDRASLI 650        660        670        680 NNAFQLVSIG KLSIEKALDL SLYLKHETEI MPVFQGLNEL 690        700        710        720 IPMYKLMEKR DMNEVETQFK AFLIRLLRDL IDKQTWTDEG 730        740        750        760 SVSERMLRSQ LLLLACVHNY QPCVQRAEGY FRKWKESNGN 770        780        790        800 LSLPVDVTLA VFAVGAQSTE GWDFLYSKYQ FSLSSTEK⁽³⁾ SQ 810        820        830        840 IEFALCRTQN KEKLQWLLDE SFKGDKIKTQ EFPQILTLIG 850        860        870        880 RNPVGYPLAW QFLRKNWNKL VQKFELGSSS IAHMNMGTTN 890        900        910        920 QFSTRTRLEE VKGFFSSLKE NGSQLRCVQQ TIETIEENIG 930        940 WMDKNFDKIR VWLQSEKLER M Protein Name Uniprot IPI Gene Name Isoform 1 of QPCT IPI00003919 Q16769-1 glutaminyl-peptide Trypsin Fragments  1. MASTPHPPG  2. YPGSPGSYA     AR     AR 10         20         30         40 MAGGRHRRVV GTLHLLLLVA ALPWASRGVS PSASAWPEEK 50         60         70         80 NYHQPAILNS SALRQIAEGT SISEMWQNDL QPLLIER² YPG 90         100        110        120 SPGSYAARQH IMQRIQRLQA DWVLEIDTFL SQTPYGYRSF 130        140        150        160 SNIISTLNPT AKRHLVLACH YDSKYFSHWN NRVFVGATDS 170        180        190        200 AVPCAMMLEL ARALDKKLLS LKTVSDSKPD LSLQLIFFDG 210        220        230        240 EEAFLHWSPQ DSLYGSRHLA AK¹ MASTPHPP GARGTSQLHG 250        260        270        280 MDLLVLLDLI GAPNPTFPNF FPNSARWFER LQAIEHELHE 290        300        310        320 LGLLKDHSLE GRYFQNYSYG GVIQDDHIPF LRRGVPVLHL 330        340        350        360 IPSPFPEVWH TMDDNEENLD ESTIDNLKNI LQVFVLEYLH L Protein Name Uniprot IPI Gene Name Isoform 1 of O75882 IPI00027235 ATRN_Human Attraction Trypsin Fragments  1. CTWLIEGQP  2. GDECQLCEV  3. GVKGDECQL     NR     ENR     CEVENR  4. LADDLYR  5. IMQSSQSMS  6. LTGSSGFVT     K     DGPGNYK  7. SCALDQNCQ     WEPR 10         20         30         40 MVAAAAATEA RLRRRTAATA ALAGRSGGPH WDWDVTRAGR 50         60         70         80 PGLGAGLRLP RLLSPPLRPR LLLLLLLLSP PLLLLLLPCE 90         100        110        120 AEAAAAAAAV SGSAAAEAKE CDRPCVNGGR CNPGTGQCVC 130        140        150        160 PAGWVGEQCQ HCGGRFR⁽⁶⁾ LTG SSGFVTDGPG NYKYKTK⁽¹⁾ CTW 170        180        190        200 LIEGQPNRIM RLRFNHFATE CSWDHLYVYD GDSIYAPLVA 210        220        230        240 AFSGLIVPER DGNETVPEVV ATSGYALLHF FSDAAYNLTG 250        260        270        280 FNITYSFDMC PNNCSGRGEC KISNSSDTVE CECSENWKGE 290        300        310        320 ACDIPHCTDN CGFPHRGICN SSDVRGCSCF SDWQGPGCSV 330        340        350        360 PVPANQSFWT REEYSNLKLP RASHKAVVNG NIMWVVGGYM 370        380        390        400 FNHSDYNMVL AYDLASREWL PLNRSVNNVV VRYGHSLALY 410        420        430        440 KDKIYMYGGK IDSTGNVTNE LRVFHIHNES WVLLTPKAKE 450        460        470        480 QYAVVGHSAH IVTLKNGRVV MLVIFGHCPL YGYISNVQEY 490        500        510        520 DLDKNTWSIL HTQGALVQGG YGHSSVYDHR TRALYVHGGY 530        540        550        560 KAFSANKYR⁽⁶⁾ L ADDLYRYDVD TQMWTILKDS RFFRYLHTAV 570        580        590        600 IVSGTMLVFG GNTHNDTSMS HGAKCFSSDF MAYDIACDRW 610        620        630        640 SVLPRPDLHH DVNRFGHSAV LHNSTMYVFG GFNSLLLSDI 650        660        670        680 LVFTSEQCDA HRSEAACLAA GPGIRCVWNT GSSQCISWAL 690        700        710        720 ATDEQEEKLK SECFSKRTLD HDRCDQHTDC YSCTANTNDC 730        740        750        760 HWCNDHCVPR NHSCSEGQIS IFRYENCPKD NPMYYCNKKT 770        780        790        800 SCR⁽⁷⁾ SCALDQN CQWEPRNQEC IALPENICGI GWHLVGNSCL 810        820        830        840 KITTAKENYD NAKLFCRNHN ALLASLTTQK KVEFVLKQLR 850        860        870        880 ⁽⁵⁾ IMQSSQSMSK LTLTPWVGLR KINVSYWCWE DMSPFTNSLL 890        900        910        920 QWMPSEPSDA GFCGILSEPS TRGLKAATCI NPLNGSVCER 930        940        950        960 PANHSAKQCR TPCALRTACG SCTSGSSECM WCSNMKQCVD 970        980        990        1000 SNAYVASFPF GQCMEWYTMS TCPPENCSGY CTCSHCLEQP 1010       1020       1030       1040 GCGWCTDPSN TGKGKCIEGS YKGPVKMPSQ APTGNFYPQP 1050       1060       1070       1080 LLNSSMCLED SRYNWSFIHC PACQCNGHSK CINQSICEKC 1090       1100       1110       1120 ENLTTGKHCE TCISGFYGDP TNGGKCQPCK CNGHASLCNT  1130       1140       1150       1160 NTGKCFCTTK ⁽⁹⁾ GVK ⁽²⁾ GDECQLC EVENRYQGNP LRGTCYYTLL 1170       1180       1190       1200 IDYQFTFSLS QEDDRYYTAI NFVATPDEQN RDLDMFINAS 1210       1220       1230       1240 KNFNLNITWA ASFSAGTQAG EEMPVVSKTN IKEYKDSFSN 1250       1260       1270       1280 EKFDFRNHPN ITFFVYVSNF TWPIKIQIAF SQHSNFMDLV 1290       1300       1310       1320 QFFVTFFSCF LSLLLVAAVV WKIKQSCWAS RRREQLLREM 1330       1340       1350       1360 QQMASRPFAS VNVALETDEE PPDLIGGSIK TVPKPIALEP 1370       1380       1390       1400 CFGNKAAVLS VFVRLPRGLG GIPPPGQSGL AVASALVDIS 1410       1420 QQMPIVYKEK SGAVRNRKQQ PPAQPGTC Protein Name Uniprot IPI Gene Name Uncharacterized IPI00925547 LTF_Human protein Trypsin Fragments  1. ADAVTLDGG  2. ARVVWCAVG  3. ARVVWCAVG     FIYEAGLAP     EQELR     EQELRK     YK  4. CAFSSQEPY  5. CFQWQR  6. CGLVPVLAE     FSYSGAFK     NYK  7. CLAENAGDV  8. CLRDGAGDV  9. CSTSPLLEA     AFVK     AFIR     CEFLRK 10. CSTSPLLEA 11. CVPNSNER 12. CVPNSNERY     CEFLR     YGYTGAFR 13. DCHLAR 14. DEYELLCPD 15. DGAGDVAFI     NTR     R 16. DGAGDVAFI 17. DLLFKDSAI 18. DLKLADFAL     RESTVFEDL     GFSR     LCLDGK     SDEAERDEY     ELLCPDNTR 19. DLKLADFAL 20. DKSPKFQLF 21. DSAIGFSR     LCLDGKR     GSPSGQK 22. DSAIGFSRV 23. DSPIQCIQA 24. DSPIQCIQA     PPR     IAENR     IAENRADAV     TLDGGFIYE     AGLAPYK 25. DVTVLQNTD 26. DVTVLQNTD 27. GEADAMSLD     GNNNEAWAK     GNNNEAWAK     GGYVYTAGK     DIK 28. GGSFQLNEL 29. GPPVSCIK 30. GPPVSCIKR     QGLK 31. GQFPNLCR   MKLVFLVLLF LGALGLCLAG RRRSVQWCA VSQPEATK⁽⁵⁾ CF QWQRNMRK VR^((29,30)) GPPV  SCIKR ^((23,24)) DS PIQCIQAIAE NR ⁽¹⁾ ADAVTLDG GFIYEAGLAP YKLRPVAAE VYGTERQPR THYYAV AVVKK⁽²⁸⁾ G GSFQLNELQG LKSCHTGLRR TAGWNVPIGT LRPFLNWTG PPEPIEAAV ARFFSA SCVPGA DK⁽³¹⁾GQFPNLCR LCAGTGENK⁽⁸⁾ C AFSSQEPYFS YSGAFK ⁽⁸⁾ CLR ^((15,16)) DGAGDVAFI RESTVFE DLSDE AER ⁽¹⁴⁾ DEYELL CPDNTRKPVDK FK⁽¹³⁾ DCHLARVP SHAVVARSV NGKEDAIWN LLRQAQE KFGK⁽²⁰⁾ D KSPKFQLFG SPSGQK ⁽¹⁷⁾ DLLFK ^((21,22)) DSAIGFSRVP PRIDSGLYL GSGYFTAIQ NLRKSEE EVAAR R^((2,3)) ARVVWCAV GEQELRKCNQW SGLSEGSVTC SSASTTEDC IALK⁽²³⁾ GEADA MSLDGGY VYTAG K ⁽⁶⁾ CGLVPVLA ENYKSQQSSDP DPNCVDRPVE GYLAVAVVR RSDTSLTWN SVKGKKS CHTAV DRTAGWNIP MGLLFNQTGSC KFDEYFSQSC APGSDPRSN LCALCIGDE QGENK^((11,12)) CV PNSNE RYYGYTGAF R ⁽⁷⁾ CLAENAGDVA FVK ^((25,26))DVTVLQN TDGNNNEAW AK^((18,19)) DLKLADF ALLCLDG KRKPV TEARSCHLA MAPNHAVVSRM DKVERLKQVL LHQQAKFGR NGSDCPDKF CLFQSET KNLLF NDNTECLAR LHGKTTYEKYL GPQYVAGITN LKK^((9,10)) CSTSPL LEACEFLRK 

We claim:
 1. A method of determining whether a subject has cancer comprising: obtaining a sample from the subject; performing steps for or detecting the level of at least one of the list of markers provided in Table 2A or Table 2B in the sample; and determining the subject has or is likely to have cancer if the levels of the marker in Table 2A are increased or if the markers in Table 2B are decreased as compared to the levels in a control sample (non-cancer).
 2. The method of claim 1, wherein the cancer is breast cancer.
 3. The method of any one of the preceding claims, wherein the sample is an ocular wash sample.
 4. The method of any one of the preceding claims, wherein the subject is a human.
 5. The method of any one of the preceding claims, wherein the markers of Table 2A are increased at least 2 fold, 4 fold, 5 fold, 8 fold, 10 fold or more relative to the level of the marker in the control sample.
 6. The method of any one of the preceding claims, wherein the markers of Table 2B are decreased at least 1.5 fold, 2 fold, 3 fold, 4 fold or more relative to the level of the marker in the control sample.
 7. The method of any one of the preceding claims, wherein a combination of markers are used to determine the likelihood of cancer in the subject.
 8. The method of any one of the preceding claims, wherein the level of the marker is detected by liquid chromatography-mass spectroscopy.
 9. The method of any one of the preceding claims, wherein the level of the marker is detected by an antibody-based detection method.
 10. The method of any one of the preceding claims, wherein the level of the marker is detected by an mRNA detection method.
 11. The method of any one of the preceding claims, wherein the subject is suspected of having cancer.
 12. The method of any one of the preceding claims, wherein the subject is at increased risk for developing a cancer.
 13. The method of claim 2, wherein the subject has a palpable lump suspected of being cancerous.
 14. The method of any one of the preceding claims, wherein the cancer is detected as a stage II breast cancer.
 15. The method of any one of the preceding claims, wherein at least three markers from Table 2A and Table 2B are used in combination.
 16. A kit for performing the method of any one of the preceding claims comprising saline solution, a collection tube and a saline collection device.
 17. The kit of claim 16, further comprising an antibody capable of binding to at least one of the markers of Table 2A or Table 2B.
 18. The kit of claim 16, further comprising a primer pair capable of amplifying at least one of the markers of Table 2A or Table 2B.
 19. The kit of any one of claims 16-18, further comprising a protease inhibitor or other protein stabilizing agent. 